It is in a natural inlet on the east side of Kings Bay and within the Crystal River National Wildlife Refuge.

[5][6] The land surrounding the springs was privately owned property until it was acquired in 2010 by a partnership between local, state and federal organizations.

[9] It "lies within the Crystal River National Wildlife Refuge and previously served as a wetland system benefitting Kings Bay".

[12] The project also used limestone boulders and native vegetation with the end result appearing to be "part of the natural landscape".

[21] A study on warm water refuge use by manatees in the Crystal River National Wildlife Refuge determined "when ambient temperatures were warmer" (>20 °C (68 °F)), the animals were "found in the Crystal or Salt Rivers or in the Gulf of Mexico" and at "colder ambient water temperatures", the manatees were "found in or near the springs (especially Three Sisters Springs)".

[5][6] However, swimmers are able to enter the spring from the water during this time with possible periodic discretionary closures due to manatee behavior.

[24] These nesting birds include anhinga, common gallinule, limpkin, mottled duck, osprey, pied-billed grebe, sandhill crane, and sora.