The significance of continuous intrarenal levels of gentamicin in the treatment of acute pyelonephritis due to Echerichia coli was investigated in rats. Treatment was started 24 hr after E. coli was injected into the left kidney. A single dose or three successive doses (10 mg/kg of body weight) of gentamicin administered ip every 8 hr could not sterilize the kidneys. Injections of gentamicin (10 mg/kg) every 12 hr for seven or 14 days resulted in continuous levels of the drug in the medulla that persisted above the minimal inhibitory concentration for E. coli (1.6 microgram/ml) for six months or more. Whereas greater than or equal to 73% of the right kidneys or urine specimens were found to be sterile up to six months following a week of therapy, only 23% of the left kidneys were sterile. Two weeks of treatment sterilized greater than or equal to 86% of the left kidneys, right kidneys, and urine specimens. Concentrations of drug in serum and urine were poor predictors of both the intrarenal distribution of drug and the outcome of pyelonephritis.