Williams is once again No. 1 on the women's side as she chases her seventh U.S. Open title. Williams also is seeking her 23rd Grand Slam singles title, which would break a tie with legendary Steffi Graf.
Djokovic is the defending men's champion and is seeking his 13th Grand Slam title. He has struggled recently due to a wrist injury and was ousted in the first round of singles play at the recent Olympics in Rio de Janeiro.
Williams has been No. 1 for 184 consecutive weeks and is vying to catch Graf's record of 186. She will open play against Ekaterina Makarova, the Russian who Williams defeated in the 2014 Open semifinals.
"I'm OK with it," Williams told reporters Friday of the matchup. "I think I try to look at it as we all always have tough matches. I played her I think in the semis before. I know she's a good player. I've just got to do the best I can."
Williams lost in the semifinals last year to unseeded Italian Roberta Vinci.
There are certainly pitfalls waiting this time around too. One of them is her fitness as Williams has been sidelined since the Olympics due to a right shoulder injury that led to her pulling out of the Western & Southern.
"I have not played a lot, I haven't practiced a lot, but I'm just now starting to feel a little better," Williams said.
Defending women's champion Flavia Pennetta of Italy retired shortly after winning the 2015 crown.
Australian Open champion Angelique Kerber of Germany drew the second seed and will face Slovakia's Polona Hercog in her first match. French Open champ Garbine Muguruza of Spain is seeded third, Poland's Agnieszka Radwanska is the fourth seed and Romania's Simona Halep is fifth.
Venus Williams landed the sixth seed. Vinci is seventh and American Madison Keys is seeded eighth.
Olympic singles champion Monica Puig of Puerto Rico is seeded 32nd.
Also, American Sloane Stephens withdrew due to an injury to her right foot.
Djokovic is trying to regain his form from the left injury that has set him back in recent matches. He will face 2013 Wimbledon semifinalist Jerzy Janowicz of Poland in his opening match.
The Serbian said he will be ready physically as he seeks his third U.S. Open title.
"I'm doing everything in my power with the medical team to make sure that I'm as close to 100 percent as possible during the course of this tournament, at least for the beginning of it," Djokovic told reporters Friday. "After undergoing certain treatments, I've gotten better.
"I'm just hoping that Monday when the tournament starts I'll be able to, as I said, get as close to the maximum of executing my backhand shot as possible."
Englishman Andy Murray landed the second seed and will face Lukas Rosol of the Czech Republic in his first match. Murray, the Wimbledon champ, won the U.S. Open crown in 2012.
Stan Wawrinka of Switzerland is seeded third and Rafael Nadal of Spain is fourth. Nadal, a two-time U.S. Open champ, has been bothered by a wrist injury.
Five-time U.S. Open champ Roger Federer isn't part of the field and is done for the season due to a knee injury.