There will be no all-Williams battle in the third round of the French Open after Venus lost to Slovakian teenager Anna Schmiedlova.
Just as world number one Serena was taking to Court Suzanne Lenglen to begin her second-round clash against Garbine Muguruza, Venus went down 2-6 6-3 6-4 on Philippe Chatrier.
The 33-year-old had looked good in beating an even younger player, 17-year-old Belinda Bencic, in round one, and started in the same vein again.
But Schmiedlova, who lost to Heather Watson in the final of the Prague Open last weekend, showed excellent composure and impressive form to turn things around.
She wobbled a little at the end, missing two match points on Venus' serve, but the American could not take her chance to level at 5-5 and Schmiedlova clinched an emotional victory.
Venus, who continues to struggle with the auto-immune condition Sjogren's syndrome, has made it past the second round at a grand slam only once in the past three seasons.
Eugenie Bouchard looked in trouble down a set to big-hitting Julia Goerges but the 18th seed recovered to win 2-6 6-2 6-1 and next meets Swede Johanna Larsson, who upset 12th seed Flavia Pennetta 5-7 6-4 6-2.