Background: Little is known about effects of factors such as kidney disease, affecting ampicillin pharmacokinetics in dogs. Objectives: Determine the pharmacokinetics of ampicillin after a single intravenous dose in healthy and azotemic dogs. Animals: Nine dogs presenting with acute kidney injury and 10 healthy dogs. Methods: This was a prospective study. An ampicillin dose of 22.2 mg/kg (mean dose) was administered once intravenously. Blood samples were obtained at timed intervals (just before administration, 1, 2, 4, 12, and 24 hours), analyzed using highpressure liquid chromatography followed by pharmacokinetic analysis of the plasma drug concentrations. Results: Peak ampicillin concentration (mcg/mL; 97.07 (36.1) vs 21.3 (50.26)), P<.001 (geometric mean (coefficient of variation, CV%)), half-life (hours; 5.86 (56.55) vs 0.97 (115.3)), P<.001) and AUC (h × mcg/mL; 731.04 (83.75) vs 33.57 (53.68)), P<.001) were greater in azotemic dogs than in healthy dogs. Azotemic dogs also had significantly lower clearance (30.06 (84.19) vs 655.03 (53.67); mL/kg h, P < .001) and volume of distribution (253.95 (30.14) vs 916.93 (135.24); mL/kg, P <.001) compared to healthy dogs. Conclusion and Clinical Importance: Increased drug concentrations and slower clearance of ampicillin in azotemic dogs could have clinical importance in contributing to antibiotic associated morbidity requiring indicating the need to adjust ampicillin dosing in dogs with decreased kidney function.