Healthy mature lactating cows (n = 6) were given gentamicin (5 mg/kg of body weight) by IV route and another dose 19 days later by IM route. Serum gentamicin concentrations were determined over a period of 48 hours after each drug dosing, using radioimmunoassay. With the aid of a nonlinear least-square regression analysis program, the combined data of the IV and IM treatments were best fitted by a 2-compartment open model, as indicated by residual trends and improvements in the sum of squares by F test and the SD of the estimated values. The distribution phase half-life was 0.25 +/- 0.12 hour, and postdistribution half-life was 1.83 +/- 0.18 hours. The volume of the central compartment was 0.10 +/- 0.02 L/kg, volume of distribution at steady state was 0.16 +/- 0.03 L/kg, and the total body clearance was 1.32 +/- 0.17 ml/min/kg. Intramuscular absorption was rapid, with a half-life for absorption of 0.63 +/- 0.28 hour. The extent of IM absorption was 92% +/- 15%. The percentage of the IM dose eliminated in the urine during the first 8 hours was 83 +/- 8. Gentamicin was detected in milk for 48 hours. Kinetic calculations predicted that IM injection of gentamicin at a dosage of 3.5 mg/kg of body weight every 8 hours would provide average steady-state serum drug concentrations of 5.08 micrograms/ml, with minimum and maximum steady-state concentrations of 1.03 and 12.05 micrograms/ml, respectively, whereas an IM injection of a 5 mg/kg dosage every 8 hours would provide average steady-state serum concentrations of 7.26 micrograms/ml, with minimum and maximum steady-state serum concentrations of 1.47 and 17.21 micrograms/ml, respectively.