An enzymatic assay was used to study the kinetics of serum gentamicin concentrations in 18 newborn infants who received doses of 0.7 to 2.4 mg/kg of gentamicin sulfate intramuscularly or in a 20-minute intravenous infusion. Maximum concentrations and half-lives were independent of gestational age, weight, hematocrit reading, and serum bilirubin and total protein values. Peak concentrations were more reproducible with intravenous infusion and correlated linearly with dose: on the average, a 1.0 mg/kg dose produced a 1.75μg/ml increment. The kinetics did not differ significantly in the 11 infants in whom the intramuscular and intravenous routes of administration were alternated. Serum half-life, urinary excretion, and volume of distribution showed marked individual variation, and differed from those reported for normal adults. These and previous results emphasize that optimal patient care requires the availability of a quantitative assay for gentamicin.