Ampicillin is commonly used to treat leptospirosis and pyelonephritis in azotemic dogs. Aminopenicillins are excreted primarily via renal tubular secretion and partially via glomerular filtration and reach high concentrations in the urine of healthy dogs, but drug clearance may be affected in dogs with kidney disease. This study evaluated plasma clearance of a single dose of ampicillin (22 mg/kg IV) in healthy dogs compared with azotemic dogs. Azotemic dogs were found to have 4.55 times higher peak ampicillin concentration, 6.04 times longer plasma half-life, and a significantly lower volume of distribution and clearance as compared with healthy dogs; however, glomerular filtration rates and creatinine levels do not correlate linearly, making dose adjustments difficult. Ampicillin clearance in azotemic dogs in this study suggests once-daily administration may be adequate for the first 48 hours of treatment; however, drug accumulation may occur after 48 hours, increasing the risk for adverse effects.