The endangered West Indian manatee Trichechus manatus has 2 recognized subspecies: the Florida T. m. latirostris and Antillean T. m. manatus manatee, both of which are found in freshwater, estuarine, and marine habitats. A better understanding of manatee feeding preferences and habitat use is essential to establish criteria on which conservation plans can be based. Skin from manatees in Florida, Belize, and Puerto Rico, as well as aquatic vegetation from their presumed diet, were analyzed for stable carbon and nitrogen isotope ratios. This is the first application of stable isotope analysis to Antillean manatees. Stable isotope ratios for aquatic vegetation differed by plant type (freshwater, estuarine, and marine), collection location, and in one instance, season. Carbon and nitrogen isotope ratios for manatee skin differed between collection location and in one instance, season, but did not differ between sex or age class. Signatures in the skin of manatees sampled in Belize and Puerto Rico indicated a diet composed primarily of seagrasses, whereas those of Florida manatees exhibited greater regional variation. Mixing model results indicated that manatees sampled from Crystal River and Homosassa Springs (Florida, USA) ate primarily freshwater vegetation, whereas manatees sampled from Big Bend Power Plant, Ten Thousand Islands, and Warm Mineral Springs (Florida) fed primarily on seagrasses. Possible diet-tissue discrimination values for 15N were estimated to range from 1.0 to 1.5 per mil. Stable isotope analysis can be used to interpret manatee feeding behavior over a long period of time, specifically the use of freshwater vegetation versus seagrasses, and can aid in identifying critical habitats and improving conservation efforts.