Since diazepam is metabolized by many organs in the rat, the microsomal fractions of the liver, kidney, and lung from male Wistar rats were assayed for NADPH-dependent metabolism of diazepam and enzymatic parameters. The predicted extraction ratios were obtained from this in vitro experimental system. The organ clearances of the liver, kidney, and lung were then calculated for the determination of the relative contribution of each eliminating organ to the total body clearance (CLtot) of diazepam in the rat. The liver was the most effective eliminating organ, followed by the kidney and the lung, in that order. The hepatic extraction ratio of diazepam was determined in vivo after portal and femoral vein administrations of diazepam. The validity of the in vitro experimental system for the liver was demonstrated by a good agreement between the calculated hepatic extraction ratio of diazepam from in vitro enzymatic parameters (0.616) and that derived in vivo (0.648). However, the sum of organ clearances of the liver, kidney, and lung did not account for CLtot of diazepam in the rat, suggesting the possible contribution of the metabolism in the other organs or tissues, or an underestimation of the pulmonary and renal metabolism.