This study characterized and compared the pharmacokinetics of piperacillin after single 100 mg/kg i.m. injections in nine red-tailed hawks (Buteo jamaicensis) and five great horned owls (Bubo virginianus) over 48 hr by a modified agar well diffusion microbial inhibition assay. The mean maximum plasma piperacillin concentrations were 204 µg/ml and 221 µg/ml for the hawks and owls, respectively, and times of maximum concentrations were 15 min and 30 min, respectively. The calculated mean terminal elimination half-lives were 77 min in the hawks and 118 min in the owls. Area-under-the-curve values were 218 ± 52 µg·hr/ml in the hawks and 444 ± 104 µg·hr/ml in the owls. On the basis of the most common minimal inhibitory concentration (90%) for various bacterial isolates from clinical samples of 8 µg/ml, analysis of the data suggests that the maximum dosing interval for piperacillin at 100 mg/kg in medium sized raptors should be 4–6 hr.