A pharmacokinetic study of ceftiofur crystalline free acid sterile oil suspension (CCFA) was performed in six apparently healthy juvenile American alligators (Alligator mississippiensis). A single intramuscular dose of 30 mg/kg was administered in the triceps muscle. Blood samples were collected prior to treatment and at 4, 12, 24, 48, 72, 120, 144, 192, 288, and 366 h post administration. Plasma samples were analyzed for ceftiofur equivalent concentrations using liquid chromatography–mass spectrometry. Pharmacokinetic parameters were determined by noncompartmental analysis. Mean peak plasma concentration was 23.2 µg/ml (range, 16.0–27.9), median time to maximum concentration was 72 h (range, 72–120), mean area under the curve from 0 to 366 h postdose was 4.24 h · mg/ml (range, 3.54–4.97), and mean terminal half-life was 143 h (range, 90.8–220). Plasma concentrations were maintained above the minimum inhibitory concentration for this study of 2.0 µg/ml, which was established from similar CCFA pharmacokinetic studies in other reptilian species, through the end of the data collection of 366 h. Because of prolonged plasma concentrations, a dosing interval could not be established in this study. Future studies should include extended collection time points and multidose studies to determine dosing regimens.