Secretary Rice, in Bucharest on the second leg of a four-nation European tour, signed the accord with Romanian Foreign Minister Razvan Ungureanu at a public ceremony in Bucharest.
"It is a remarkable thing to think that some 16 years after the collapse of communism here in Romania... we would be signing an agreement that will allow America to have forces" in Romania, Ms Rice said.
"It is fitting that the United States and Romania are not just friends but our forces are brothers and sisters in arms," she said, adding: "We know we have a great and committed partner in Romania."
Romania has 860 troops with the US-led coalition in Iraq, as well as soldiers in Afghanistan. Romanian President Traian Basescu said his country would stay with the Americans "to the end" of the mission.
Rendition claims
Ms Rice's trip, which took her to Germany beforehand, has been dominated by outcry over alleged CIA flights through European states, reportedly carrying terrorism suspects to secret prisons.
Poland and Romania have been mentioned as countries hosting detention centres where torture was allegedly carried out.
But President Basescu said the reports were "based only on speculation", reiterating earlier Romanian denials.
Mr Basescu said planes had landed in Romania as part of collaboration with the US in the "political and military domains, but also in secret service work".
"But it is wrong to say, based on information from take-offs and landings that Romania hosts secret prisons or practices torture," he insisted.
Human Rights Watch has said Romania may have hosted a secret prison at the Mikhail Kogalniceanu Air Base, where the new US military presence will be headquartered.
The centre for the so-called Eastern European Task Force (EETAF) will be at the air base near Constanta on the Romanian Black Sea coast and will house about 1,500 American troops who will train with Romanian soldiers, a US State Department official said.
US forces will be also using three other sites in Romania – Smardan training range, Babadag training area and Cincu training range – Mr Basescu said.
Torture denied
In Berlin on Tuesday Secretary Rice said that the United States "does not condone torture", as it was against US law and its international obligations.
Before leaving Washington, Ms Rice went on the counter-attack, insisting tough tactics were needed to fight terrorism and suggesting its allies knew more than they were saying.
"You can't prevent an attack if you don't know about it," she reiterated in Bucharest.
"Intelligence is the only way you know about an attack before one is actually to take place," she said.
Ms Rice left Bucharest for Kiev on Tuesday evening. The final leg of her five-day trip will take her to Brussels for a NATO meeting.