Serena and Venus Williams saw hopes of one last US Open glory run together scuppered by a Czech pair who apologised for knocking out the American superstars.

The Williams sisters lost 7-6 (7-5) 6-4 against Lucie Hradecka and Linda Noskova on Arthur Ashe Stadium, with the doubles match given top billing in the New York night session.

Such scheduling had previously been unheard of, but such is the attention being foisted on the sisters that first-round doubles encounter was the match everyone wanted to see, knowing it could well be the last time the siblings play as a team.

Serena has indicated, albeit couched in somewhat vague terms, that this US Open will be her farewell to tennis. Venus, now 42, may follow suit and also retire from the sport.

While 40-year-old Serena remains in the singles, having beaten world number two Anett Kontaveit in round two to set up a Friday night clash with Ajla Tomljanovic, the dream of one more Flushing Meadows doubles title is over.

It was Czech-out time for the duo who have won 14 grand slam doubles titles together, and Hradecka said sorry to the partisan crowd for spoiling the party.

"I'm still in shock that we won, because we were playing for the first time with each other and I think we did a very good job," Hradecka said.

"I'm so sorry for you that we beat them, but we are so happy that we did it."

Hradecka had been planning on playing with Indian Sania Mirza, but an injury for Mirza put paid to that idea, meaning a new partner was required.

Noskova, 17, answered the 37-year-old Hradecka's call, and enjoyed a night to remember.

"Playing against the Williams sisters is a special moment for everybody, anytime," Noskova said.

"I was really lucky I could play with my doubles partner and we could win and pull out a good fight."

It was the Williams' first doubles match at a grand slam since the 2018 French Open, and their first at the US Open since 2014, and although it was a tight contest the younger pair just had the edge.

Neither sister gave an on-court interview or a press conference after the match, with all eyes now turning towards Serena's singles campaign, and the possibility of that extending deep into the second week.