The pharmacokinetics of gentamicin C₁, C₂, and C_1a 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, C_1a, and C₂. The mean residence times of gentamicin C₁, C_1a, 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 min¹ kg¹, was significantly higher (P = 0.0156) than CL of gentamicin C_1a, 1.81 ± 0.26 ml min¹ kg¹, and C₂, 1.82 ± 0.25 ml min¹ kg¹. Similarly, the volume of distribution at steady state (V_ss) of gentamicin C₁, 0.36 ± 0.04 liter kg¹, was significantly higher (P = 0.0156) than the V_ss of gentamicin C_1a, 0.14 ± 0.01 liter kg¹, and C₂, 0.15 ± 0.02 liter kg¹. Tissue binding was considered the most likely cause for the difference. The difference may have clinical and toxicological significance.