The pharmacokinetics of gentamicin C1, C2, and C1a were studied in six beagles after administration of gentamicin at 4 mg/kg of body weight as a single intravenous bolus dose. Plasma concentrations of the gentamicin components were analyzed with a novel high-performance liquid chromatography method capable of identifying and quantifying each of the components. The pharmacokinetic analysis of the plasma concentration-versus-time data was performed using the noncompartmental approach. The results indicated significant differences in the pharmacokinetic characteristics between the gentamicin components C1, C1a, and C2. The mean residence times of gentamicin C1, C1a, and C2 were 81 ± 13, 84 ± 12, and 79 ± 13 min (mean ± standard deviation), respectively. The half-lives of the respective components were 64 ± 12, 66 ± 12 and 63 ± 12 min. Clearance (CL) of gentamicin C1, 4.62 ± 0.71 ml min−1 kg−1, was significantly higher (P = 0.0156) than CL of gentamicin C1a, 1.81 ± 0.26 ml min−1 kg−1, and C2, 1.82 ± 0.25 ml min−1 kg−1. Similarly, the volume of distribution at steady state (Vss) of gentamicin C1, 0.36 ± 0.04 liter kg−1, was significantly higher (P = 0.0156) than the Vss of gentamicin C1a, 0.14 ± 0.01 liter kg−1, and C2, 0.15 ± 0.02 liter kg−1. Tissue binding was considered the most likely cause for the difference. The difference may have clinical and toxicological significance.