The edict has already sparked a row within the Roman Catholic Church and has been widely condemned by gay and civil rights groups when leaked extracts were published by newspapers last month.
The prohibition applies to seminarians involved in homosexual practices as well as those displaying "deep-seated homosexual tendencies" or those who support "gay culture."
The Church "while profoundly respecting the persons in question, cannot admit to the seminary or to holy orders those who practice homosexuality, present deep-seated homosexual tendencies or support the so-called 'gay culture.'"
"One must in no way overlook the negative consequences that can derive from the ordination of persons with deep-seated homosexual tendencies," the 21-paragraph instruction says.
Sex scandals
The instruction follows a series of sex scandals within the Catholic Church in the United States, Latin America and Europe, and belated Vatican efforts to tackle priestly paedophilia.
However, the edict approved by the 78-year-old pope leaves the door to the priesthood ajar for men whose homosexuality was only "the expression of a transitory problem -- for example that of adolescence not yet superseded."
It said men undergoing such a passing phase of homosexuality must show their tendencies had been "clearly overcome at least three years before ordination to the diaconate." A seminarist becomes a deacon up to two years before full ordination.
The document does not say how a candidate would be able to prove that his "transitory problem" had been overcome, or by what means it could be independently determined.
The Vatican began sending teams in September to inspect more than 200 seminaries in the United States with a mandate to look for "evidence of homosexuality."
The idea was first mooted after the late Pope John Paul II summoned US bishops to the Vatican in 2002 to discuss a spate of sex scandals across the US, mostly involving paedophile priests, but that in turn led to allegations that the Vatican was confusing homosexuality with paedophilia.
The US bishops' conference had then publicly conceded there was a "homosexual atmosphere" in many seminaries.
Document leaked
The leak last month of the document published on Tuesday provoked outrage among many Catholic groups in Europe and the United States.
One dissident Catholic newsletter, Adista, described the move as "ethical cleansing."
The Vatican unsurprisingly has no official estimate of the number of homosexual men in the ranks of the priesthood.
However, Donald Cozzens, a priest and author of "The Changing Face of the Priestood," estimates that at least 25 percent of men in seminaries and the priesthood in the US alone are gay.
The edict contained "an element of hypocrisy," he said in the wake of last month's leaks, adding that a religious superior tasked with informing a gay candidate he could not proceed to the priesthood would, in many cases, be gay himself.
David and Jonathan, a French organisation of gay Catholics, dismissed the edict as "against truth but above all, reckless, inappropriate and dangerous."
New Ways Ministry, a group of US priests working with homosexuals, said it feared the instruction displayed "a woefully narrow and ignorant understanding of homosexuality" and would cause "deeper personal and spiritual damage" to gay priests and seminarians.
Leftwing Italian deputy Franco Grillini, honorary president of the Italian gay association Arcigay, has slammed the edict as "discrimination bordering on racism."
The document was approved by Pope Benedict on August 31 -- during his vacation at his summer residence in Castelgandolfo -- and published by the Vatican's Congregation for Catholic Education, which has responsibility for running seminaries.
Limbo to go
Meanwhile the days of Limbo, the place the Catholic Church says unbaptised babies go, may be numbered.
According to Italian media reports, an international theological commission will advise Pope Benedict to eliminate the teaching about limbo from Catholic catechism.
The Catholic Church teaches that babies who die before they can be baptised go to limbo, whose name comes from the Latin for "border" or "edge", because they deserve neither heaven nor hell.
Last October, seven months before he died, Pope John Paul asked the commission to come up with "a more coherent and enlightened way" of describing the fate of such innocents.
The commission, which has been meeting behind closed doors, may make its recommendation soon.