"My goal and hope was to play in this tournament. Unfortunately, in my last training session yesterday, I felt a sharp pain in my leg again," the 31-year-old Spanish star said hours before what was to have been his opening match of the ATP Tour event in Acapulco.
Nadal, favoured to win the tournament and champion here in 2005 and 2013, had been due to face fellow Spaniard Feliciano Lopez.
"I took all the appropriate steps to arrive at the tournament in form. I went to Cozumel first, to adapt (to the climate)," he told reporters.
Nadal limped out of the quarter-finals at the Australian Open on January 23. He has since lost his number one ranking to Roger Federer.
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Nadal said doctors in Mexico warned him not to play for fear of aggravating the injury.
"I still don't know what it is, because we don't know. It seems it's not as bad as what I had at the Australian Open," he said.
"Now, my main goal is to find out the extent of the injury."
- Injury unclear -
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The 16-time Grand Slam winner Nadal previously announced himself fully fit and said he was looking forward to competing in his second event of 2018.
"I took a couple of weeks of rest and worked hard on rehabilitation," he said earlier in the week. "Last week, I started practising hard again. I feel ready."
Nadal is expected to remain at least one more day in Acapulco to have more tests done.
While a victory this week would not have been good enough to overhaul Federer, it would leave Nadal primed to replace the Swiss star at Indian Wells or Miami next month.
The 2008 Olympic gold medal winner is known as the "King of Clay" and is considered the greatest clay-court player in the history of the sport.
But his success is not limited to clay. Nadal has won 10 French Open titles, three US Opens, two Wimbledons and one Australian Open.
In men's action on Tuesday, defending champion Sam Querrey lost his opening match and is gone from the tournament after he was beaten by Australia's Matthew Ebden 6-3, 1-6, 7-6 (7/5).
Querrey, who was seeded seventh, hit nine aces but had six double faults and won just 38 percent of his second-serve points.
Juan Martin Del Potro, who returned to the top 10 ranking last month for the first time in three years, clobbered Mischa Zverev 6-1, 6-2 in just 61 minutes.
He advances to the second round where he will face Spain's David Ferrer. This will be the 12th career meeting between the two with Ferrer holding a 6-5 edge.
Canadian Denis Shapovalov rallied to beat Kei Nishikori of Japan 6-7 (3/7), 6-3, 6-1 in a two hour, 17 minute marathon match.
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