The large mammals swam into Three Sister Springs in Kings Bay, forcing an acre of the area to close.
Officials were forced to shut a spring in Florida after an estimated 300 manatees gathered in the warm water.
The mammals, which looked like large rocks beneath the surface, swam into Three Sister Springs in Kings Bay in Crystal River, shutting down an acre of the area on Monday afternoon and pushing out swimmers and kayakers.
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The spring was expected to open Tuesday but it will close again when the animals return during high tide.
"Thank you for your understanding and patience as we allow the manatees their space to rest and stay warm," Crystal River National Wildlife Refuge Complex wrote on Facebook.
Experts say the manatees use the area as a warm water shelter during the winter.
Manatees suffer hypothermia in waters colder than 65 degrees but the dozens of springs in Kings Bay have a constant temperature of 72 degrees, Miles Saunders of the Citrus County Visitor and Conventions Bureau told INSIDE EDITION.
"This is the most important winter refuge for manatees in the world," he said.
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Last month, the U.S. Fish and Wildlife Service reported a record 1,047 manatees in Citrus County, he said. There are around 6,000 manatees across the entire state.
The average adult manatee measures around 10 feet long and can weigh as much as 1,200 pounds. They are known as gentle and slow animals who do not pose a danger to humans, but staff at Crystal River encourages swimmers to keep their distance and respect the creatures' space.
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