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Objective: Gentamicin is an important factor in the empiric therapy of premature babies with suspected invasive bacterial infection. The aims of the study were to assess the tolerability of short course of gentamicin in preterm neonates., Patients and Methods: Preterm neonates aged 24 hours or less who were born at 32-37 weeks of gestation and weighed over 1500 grams were included in the study. Those infants suspected of having invasive non-CNS bacterial infection were assigned to treatment with ampicillin 50 mg/kg twice daily and either ODD (once daily dosing) or twice daily dosing (TDD) of gentamicin 5 mg/kg/day (17 and 18 patients, respectively). Neonates with shock, impaired renal function and known kidney, ear, and heart malformations, and metabolic disease were excluded from the study. At 72 to 96 hours of therapy, serum and urine creatinine, and sodium concentrations, peak (PGt) and though (TGt) serum levels of gentamicin, and urinary lysosyme secretion were measured. Fractional excretion of sodium (FeNa), and glomerullar filtration rate (GFR) were calculated for each infant. Audiometric evaluation was performed at 1 to 2 months of age., Results: For the ODD and TDD groups respectively, the values of serum creatinine, FENa, GFR, and urinary lyzozim were similar. The mean SD PGt levels were 9.9 +/- 4.6 vs 5.9 +/- 1.9 g/ml (p < 0.04), and that of the TGt levels was 1.55 +/- 0.55 vs 2.4 +/- 0.9 micrograms/ml (p = 0.028), Ten (55.6%) vs 3(23.1%) of the TDD and ODD groups respectively had TGt levels above 2 micrograms/ml (p = 0.035). Audiometric evaluation was normal in all infants., Conclusions: Short course of ODD of gentamicin could be safe and potentially more effective in preterm babies.