The 30th career meeting between Serena and Venus also marks the first time the sisters have faced each other this early at a Grand Slam since their first meeting at a major -- the second round of the 1998 Australian Open.
Venus is the 16th seed, one place above her sister who is still working her way back up the rankings after returning from maternity leave earlier this year.
Serena, gunning for a record seventh title at the U.S. Open, was beaten by Venus in their most recent meeting at Indian Wells but won their last Grand Slam encounter in the final of the 2017 Australian Open to secure her 23rd Grand Slam title.
Stephens is up against hard-hitting Victoria Azarenka while Nadal, who eased through his opening rounds with a minimum of fuss, takes on Russian Karen Khachanov, the number 27 seed.
Argentine Juan Martin del Potro is also in action, taking on Spaniard Fernando Verdasco. Del Potro, the third seed, is yet to drop a set in New York so far and saw off American duo Donald Young and Denis Kudla in the first two rounds.
Fifth seed Kevin Anderson of South Africa, who was runner-up last year, will get his first action on one of the main courts when he takes on Canada's Denis Shapovalov at the Louis Armstrong Stadium.
Shapovalov's compatriot Milos Raonic, who is seeded 25th, is up against 2016 champion and wild card Stan Wawrinka, while ninth seed Dominic Thiem of Austria will face American Taylor Fritz.
(Reporting by Simon Jennings; Editing by Peter Rutherford)
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