Enrofloxacin is a fluoroquinolone antibiotic that is effective against many of the common gram-negative bacterial pathogens in raptors. This 3-period crossover study was designed to compare the pharmacokinetics of enrofloxacin after oral, intramuscular, or intravenous administration in red-tailed hawks (Buteo jamaicensis; n = 8) and great horned owls (Bubo virginianus; n = 5). In each study period, birds received a single dose of an injectable formulation of enrofloxacin (15 mg/kg) by 1 of these 3 routes. Oral administration was accomplished by force-feeding each bird a small freshly killed mouse that had received an intraperitoneal injection of enrofloxacin (in-prey). Intramuscular injections were divided into 2 sites in the pectoral musculature. Serial plasma samples were taken before administration and at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 hours after administration. Oral administration resulted in plasma concentrations of enrofloxacin that peaked at 4–8 hours in both species. Plasma concentrations remained at >1 μg/ml until at least 18 hours after administration, although an initial lag time of approximately 2–8 hours occurred for absorption from the gastrointestinal tract. Intramuscular administration resulted in plasma concentrations that peaked at 0.5–2 hours; enrofloxacin levels remained at >1 μg/ml for at least 15 hours. After intravenous administration to red-tailed hawks, enrofloxacin levels remained at >1 μg/ml for at least 15 hours. Two great horned owls showed acute weakness, bradycardia, and peripheral vasoconstriction during intravenous injection. These clinical signs resolved within 1–3 hours with supportive therapy; however, the intravenous route was not evaluated for the other 3 owls. We conclude that oral (in-prey) and intramuscular routes are reliable means of administration of injectable enrofloxacin in red-tailed hawks and great horned owls, using a dosage of 15 mg/kg q24h. Intravenous administration of enrofloxacin can be performed with caution in red-tailed hawks but should not be attempted in great horned owls.