Marketa Vondrousova's Wimbledon title defence came to an early end as she fell to a surprise first-round defeat by Spain's Jessica Bouzas Maneiro.
The Czech became the first unseeded player to win the women's singles trophy when she triumphed against Ons Jabeur in last year's final.
But Vondrousova's game was riddled with unforced errors and double faults as the 25-year-old opened proceedings with a lacklustre performance on Centre Court.
World number 83 Bouzas Maneiro, 21, took advantage on Tuesday to win 6-4 6-2 for her first Grand Slam victory.
It is the first time since 1994, when Steffi Graf lost to Lori McNeil, that the women's defending champion has lost in the first round at Wimbledon.
"I am really happy, I think this is one of the most important moments in my life, in my career," said Bouzas Maneiro.
"This is the most beautiful tournament I have played in my life so thank you to everyone who came to watch the match today."
Vondrousova's early exit means there will be a different women's singles champion for a seventh consecutive year and she is the first women's defending champion in 30 years to lose in the first round.
Serena Williams was the last player to successfully defend the Venus Rosewater Dish, winning back-to-back titles in 2015 and 2016.
There was no concern for world number one Iga Swiatek who secured a comfortable 6-3 6-4 victory over 2020 Australian Open champion Sofia Kenin.
The American had her serve broken in her opening service game by the Polish 23-year-old, who has already won five Grand Slam titles but never made it beyond the quarter-finals at Wimbledon.
In round two, Swiatek will play Petra Martic of Croatia after she got past British wildcard Fran Jones in three sets.
Meanwhile, Caroline Wozniacki, the 2018 Australian Open champion, dispatched world number 40 Alycia Parks 6-2, 6-0 in just 53 minutes.
The 33-year-old Dane has never got past the fourth round at Wimbledon but looked comfortable against her American opponent.
Last year's beaten finalist Ons Jabeur also breezed through, winning her match against Japan's Moyuka Uchijima 6-3 6-1 in 55 minutes.
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