The defeat came after a closely contested match against Taiwanese Chan Hao-Ching and Australian Maya Joint, which ended in a 2-1 loss for the Americans. Both the second and third sets went to tiebreaks.
It is the first time that Serena, 44, and Venus Williams, 46, have played together in the tournament since 2022. The announcement that the sisters had received wildcards for the US Open came at the end of August.
The Williams sisters have previously won the doubles tournament in 1999 and 2009.
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