Since noting an apparent increase in hypocalcemia in neonates receiving gentamicin every 24 h (q 24 h) for > or = 4 days, we have prospectively monitored serum calcium (Ca) values in these patients receiving prolonged gentamicin therapy. This study is a retrospective analysis of those values measured during gentamicin treatment. The study included neonates with gestational age > or = 35 weeks who received > or = 4 days of gentamicin therapy and in whom at least one serum Ca value was measured > or = 47 h after initiation of therapy. Hypocalcemia was defined as a serum Ca level < 8 mg/dl (2 mmol/l). Data were analyzed by Student t-test, chi-square test, and Pearson product moment correlation. There were 1,624 neonates that met the study criteria. Ca was < 8 mg/dl in 241 (15%). Ca < 8 mg/dl was more likely in boys than in girls (16.4% vs 11.8%, P = 0.01) and in neonates < 37 weeks gestational age (GA) than in those > or = 37 weeks GA (23.9% vs 14.1%, P = 0.01). A second Ca value was obtained in 883 neonates (54%); 23.2% of neonates with initial Ca < 8 mg/dl remained hypocalcemic, and 30% of these were receiving oral Ca supplementation. The second Ca value was < 8 mg/dl in eight neonates in whom initial Ca was > or = 8 mg/dl. Hypocalcemia is not uncommon in neonates receiving gentamicin therapy, and it may occur more frequently in boys and late-preterm infants. These data suggest that the monitoring of serum Ca levels should be considered when gentamicin is given > or = 4 days.