Dosing regimens often recommend lower gentamicin doses in neonates (3-5 mg/kg) than in older children (7 mg/kg or more) despite the higher volume of distribution in neonates. We studied an extended-interval high-dose (6 mg/kg) gentamicin regimen in a single tertiary neonatal unit from 2004-2012. During the first week of life, dosing interval was 24 h for term infants, 36 h for preterm infants with gestational age (GA) 29-36 weeks and 48 h for preterm infants with GA <29 weeks. After the first week of life, dosing interval was 24 h if corrected age (GA + postnatal age) ≥29 weeks and 36 h if corrected age <29 weeks. Outcome measures were trough plasma concentration (TPC), ototoxicity and prescription errors. In 546 treatment episodes, TPC was measured prior to the third gentamicin dose. There were 37 episodes (6.7 %) of prescription errors, mainly a too long dosing interval. We included 509 treatment episodes (440 infants) in the final analysis. Mean (standard deviation) gentamicin TPC during the first week of life was 1.1 (0.5) mg/L and after the first week of life 0.8 (0.6) mg/L. In 31 (6 %) episodes, TPC was ≥2.0 mg/L, predominantly among term infants with renal impairment. Thirty-eight patients failed the neonatal hearing screening, but only four of these 38 had permanent hearing loss. All four had a TPC <2.0 mg/L. Conclusions: This extended-interval high-dose gentamicin regimen was associated with low numbers of elevated TPCs, low numbers of prescription errors and no evidence for ototoxicity.