Recent studies have suggested the inadequacy of an initial gentamicin 2.5 mg/kg standard dose in neonates and the need for a loading dose. The purpose of this prospective, randomized study was to compare initial peak and initial trough serum gentamicin concentrations (SGC) in neonates after a standard dose (2.5 mg/kg) or a loading dose (4 mg/kg) on the first day of life. A secondary objective of the study was to evaluate the use of two SGC drawn after the first dose in designing individualized dosage regimens, despite the many changes in gentamicin disposition that occur over the first week of life. Forty infants admitted to the NICU were randomized to receive either 2.5 or 4 mg/kg gentamicin. Individual gentamicin pharmacokinetic parameters were determined after the first dose. Initial peak SGC were > 5 mcg/ml in only 6% of neonates receiving 2.5 mg/kg, versus 94% of neonates receiving 4 mg/kg. The initial trough after the first dose was < 2 mcg/ml in 100% of patients receiving 2.5 mg/kg and only 39% of patients receiving 4 mg/kg. Using two SGC after the first dose successfully predicted steady state peaks in 13/16 infants and steady state troughs in 14/16 infants. Thus, standard treatment of 2.5 mg/kg gentamicin yields initial peak serum gentamicin concentrations < 5 mcg/ml in neonates while a 4 mg/kg gentamicin loading dose, combined with pharmacokinetic monitoring after the first dose, optimizes gentamicin therapy in neonates.