(CNN) - They were one of the biggest favorites for gold -- but Serena and Venus Williams won't be topping the podium together at the Rio 2016 Olympics.
The sisters, who won the women's doubles title in 2000, 2008 and 2012, were stunned in the first round by the Czech pairing of Lucie Safarova and Barbora Strycova.
Seeded first at the tournament, the Americans were beaten 6-3 6-4 in 93 minutes -- their first ever doubles defeat at the Games in 16 matches.
This defeat capped a couple of miserable days for older sibling Venus, who was dumped out of the singles draw in the first round by Belgium's Kirsten Flipkens.
Earlier Sunday, Serena started the defense of her singles title with a 6-4 6-2 win over Australia's Daria Gavrilova.
She will play Alize Cornet in the second round after the French player overcame Sweden's Johanna Larsson.
No. 2 seed Angelique Kerber of Germany also progressed after defeating Colombia's Mariana Duque-Marino.
LIVE: Day two of the Olympic Games
In the men's draw, defending champion Andy Murray set up a second round clash against Argentina's Juan Monaco after cruising to a 6-3 6-2 win over Serbia's Viktor Troicki.
He is joined by 2008 champion Rafael Nadal, who overcame Argentina's Federico Delbonis in straight sets to book a date with Italy's Andreas Seppi.