The pharmacokinetics of gentamicin were studied in six healthy mature horses of mixed breeding and of both sexes. A parenteral preparation of gentamicin sulfate (5% aqueous solution) was administered rapidly (IV) at the dosage level of 5 mg/kg of body weight. Venous blood samples were taken at 0 (base line), 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, and 120 hours after gentamicin administration. Serum gentamicin was measured by a radioimmunoassay technique. The gentamicin concentration data was fitted to a one- and two-compartment open model with first-order elimination from the central compartment with the aid of nonlinear least squares program. The data were found to be best described by the two-compartment model with r2 = 0.997. Half-life, as determined from the terminal phase was 2.54 +/- 0.33 hours. Calculation of the total body clearance provided a mean of 1.16 +/- 0.11 ml/minute/kg of body weight (1.04 to 1.31 ml/minute/kg, range); the volume of distribution calculated from the area under the curve was determined separately for each animal and had a mean value of 0.254 +/- 0.036 L/kg. The initial exponential decline (alpha) in gentamicin serum concentration had an average value of 3.75 +/- 1.86 hours-1, whereas the terminal values were described by beta = 0.275 +/- 0.036 hours-1. Other pharmacokinetic values determined also are presented.