A standard gentamicin dosage regimen intended to result in fewer trough concentrations of >2 microg/mL was studied. At a neonatal intensive care unit, gentamicin dosage guidelines of 2.5 mg/kg (as the sulfate) administered i.v. over 30 minutes every 12, 18, or 24 hours to neonates with a gestational age (GA) of > or =30 weeks were resulting in some relatively high trough serum concentrations (>2 microg/mL). Pharmacokinetic values derived for this baseline group were used to predict the gentamicin concentrations that would result from a standard regimen of gentamicin 3.5 mg/kg i.v. over 30 minutes every 24 hours. No patient in the baseline group was predicted to have a trough of >2 microg/mL with the new regimen, which was then approved for routine use. The new regimen was used for every neonate with a GA of > or =30 weeks who was admitted and treated with gentamicin (the protocol group). One set of concentrations was collected for each infant. Compared with the baseline group, the protocol group had significantly lower trough and significantly higher peak gentamicin concentrations. The total frequency of high troughs in the baseline group (23 [33%] of 69 patients) differed significantly from that in the protocol group (3 [4%] of 74 patients). No patient had or developed renal impairment. A gentamicin dosage protocol of 3.5 mg/kg every 24 hours for neonates with a GA of > or =30 weeks resulted in higher gentamicin peaks, lower troughs, and a lower frequency of troughs of >2 microg/mL, compared with previous dosage practice.