There is relatively little information available on how the relative importance of fresh, brackish, or marine environments may vary geographically or individually with respect to manatee Trichechus manatus latirostris feeding ecology. As a first application of stable isotope analysis to determine diet composition of wild manatees, skin from 25 manatee carcasses and leaf tissue from 25 plant species were collected from 4 regions in Florida and analyzed for relative values of stable carbon (δ 13 C) and nitrogen (δ 15 N) isotopes. Values of δ 13 C and δ 15 N measured in plants ranged from -0.8 ± 1.0 to 6.4 ± 0.0‰ and in manatee tissues from -8.7 ± 0.2 to -28.3 ± 0.1‰, respectively. A mix- ing model was applied to estimate manatee diet composition across different geographic regions, and results (mean, all manatees, all regions) indicated that, overall, Florida manatees consumed 44% of their diet from marine and/or estuarine sources. Manatees living along the central east coast obtained 100% of their diet from marine and/or estuarine sources, while animals recovered from the St. John's River system (NE region) obtained ~50% of their diet from freshwater sources, suggesting that some time had been spent outside of the river basin. In conclusion, results of the stable isotope analysis suggest that, even though manatees have a physiological requirement to ingest fresh water, marine and estuarine environments supply a significant fraction of their food resources.