The present study investigated the effects of melatonin, an antioxidant, on gentamicin-induced nephrotoxicity in rats. Melatonin (5 mg/kg p.o.) was used 3 days before and 8 days simultaneously with gentamicin (80 mg/kg i.p.) Saline-treated animals served as controls. Determinations of urinary creatinine, N-acetyl-β- D-glucosaminidase, glucose, protein, blood urea, serum creatinine, plasma and kidney tissue malondialdehyde (MDA), and antioxidant enzyme levels in kidney tissue were done after 8 days of gentamicin treatment. The kidneys were also examined for morphological changes using histological techniques. Gentamicin caused nephrotoxicity as evidenced by marked elevation in blood urea and serum creatinine. Mean blood urea and serum creatinine levels were 289 ± 50, and 2.5 ± 0.5 mg/dl, respectively, in rats treated with gentamicin. Melatonin significantly protected the rats from gentamicin-induced nephrotoxicity; blood urea and serum creatinine levels were 23 ± 2.7 and 0.88 ± 0.19 mg/dl, respectively. The creatinine clearance was decreased with gentamicin treatment (0.048 ± 0.007 ml/min) as compared with controls (0.41 ± 0.08 ml/h/kg). In rats treated with melatonin plus gentamicin, the creatinine clearance was similar to controls (0.41 ± 0.08 ml/h/kg). The product of lipid peroxidation (MDA) was markedly increased in plasma (2.10 ± 0.15 nmol) and kidney tissue (8.87 ± 3.2 nmol/mg protein) with gentamicin treatment. Melatonin prevented the gentamicin-induced rise in plasma MDA (1.03 ± 0.27 nmol) and kidney tissue MDA (2.57 ± 0.87 nmol/mg protein). An increased excretion of urinary N-acetyl-β- D-glucosaminidase, glucose, and protein by gentamicin was also prevented by melatonin. Kidneys from gentamicin-treated rats showed tubular epithelial loss with intense granular degeneration involving more than 50% of renal cortex, while there were findings comparable to controls in melatonin plus gentamicin treated rats. The present study indicates that melatonin significantly protects against gentamicin-induced renal toxicity in Wistar rats.