OBJECTIVE To determine the serum levels of gentamicin in newborn on one or two daily doses. METHODS Retrospective and subsequently prospective, descriptive. METHODS Department of Pediatrics of the Deventer Hospital, the Netherlands. METHODS Gentamicin trough and peak plasma levels were analysed retrospectively in 30 neonates to whom gentamicin 3-4 mg/kg/day in two doses had been administered in 1995. In a subsequent prospective study in 1996, 52 neonates received gentamicin 4 mg/kg/day in one dose. Optimal levels were defined as a trough of < 2 mg/l and a peak of 6-12 mg/l. Levels were determined using a immunoassay. RESULTS Both groups were similar in gestational age, weight and age at the start of treatment. Both trough and peak plasma levels were not known in all patients. Adequate gentamicin trough levels were seen more often in the 'once daily' group than in the 'twice daily' group, in both the premature (12/14 = 86% versus 3/7 = 43%; p = 0.04) and non-premature (36/37 = 97% versus 18/23 = 78%; p = 0.017) neonates. Peak levels were higher in the 'once daily' group than in the 'twice daily' group, in both the prematures (11/13 = 85% versus 1/7 = 14%; p = 0.002) and non-prematures (25/28 = 89% versus 2/22 = 9%; p < 0.000001). Optimal trough and peak levels were found in 9/13 (69%) of the premature and 24/27 (89%) of the non-premature neonates treated according to the 'once daily' schedule, compared with 0 and 1/22 (5%) respectively in the neonates treated according to the 'twice daily' regimen (p = 0.003 and p < 0.0001, respectively). CONCLUSIONS In view of the reported levels and the literature, an initial dose of gentamicin of 4.0 mg/kg/day once a day is advised for neonates.