Penicillin G, penicillin V, ampicillin, hetacillin, and amoxicillin were administered separately per os to clinically normal adult dogs of both sexes at 8-hour intervals for five consecutive 8-hour test periods. All urine was collected from each dog during each test period and was assayed for antimicrobial activity. Daily doses of the antimicrobics used were as follows: penicillin G, 110,000 U/kg of body weight; penicillin V, 77 mg/kg; ampicillin, 77 mg/kg; hetacillin, 77 mg/kg; and amoxicillin, 33 mg/kg. Mean 8-hour urine concentrations (+/- 1 SD) were as follows: penicillin G, 294 +/- 210 U/ml; penicillin V, 148 +/- 98 micrograms/ml; ampicillin, 309 +/- 55 micrograms/ml; hetacillin, 300 +/- 156 micrograms/ml, and amoxicillin 201 +/- 93 micrograms/ml. Among the three antimicrobials administered at the same daily dosage, ie, penicillin V, ampicillin, and hetacillin, the mean urine concentrations of penicillin V were significantly lower (P < 0.01) than were concentrations of ampicillin and hetacillin. The mean urine concentrations of the latter two did not differ significantly (P > 0.05).