Drug-induced nephrotoxicity (NT) has become an increasingly significant clinical problem. An in vitro model of drug-induced NT was therefore developed using gentamicin and the effects of ATP-MgCl2 on reduction or prevention of NT were determined. To study this, non-pulsatile perfusion in isolated rat kidneys was maintained at 100 mm Hg during 2 hr of perfusion at 37°C. The oxygenated Krebs-HCO3 perfusate contained 7.5 g/dl albumin as colloid, glucose, creatinine, amino acids, trace amounts of [3H]inulin and 125I-lysozyme, and either 0, 0.4, 0.8, or 1.2 mg/ml of gentamicin. In some studies, 2 mM ATP-MgCl2 was added with 0.8 mg/ml of gentamicin at 0 and 60 min of perfusion. During each 10-min clearance period, glomerular filtration rates, sodium absorption, water absorption, and fractional clearance of TCA-precipitable lysozyme were measured. The results indicate that renal perfusate flow, glomerular filtration rate, urinary flow and tubular absorption of protein (a sensitive indicator of tubular function), sodium, and water were affected by gentamicin in a dose-dependent manner. An isolated kidney preparation can therefore be used to study gentamicin-induced NT. Higher in vitro perfusate concentrations of the drug were needed, however, to acutely mimic the in vivo cumulative effects. Nonetheless, renal perfusate flow, glomerular filtration rate, and the depression in protein reabsorption which occurred with gentamicin treatment were markedly improved by simultaneous treatment with ATP-MgCl2. Thus, ATP-MgCl2 may be useful in reducing drug-induced nephrotoxicity. © 1985.