Acute ethanol intake has been shown to decrease plasma testosterone levels in rodents and man. This is mainly due to inhibition of testosterone synthesis, but there is still some controversy about the mechanisms responsible for the inhibition. These mechanisms were studied in the present experiments with isolated rat Leydig cells. Leydig cells were incubated for two hours in sealed plastic vials with various concentrations of ethanol (0.3-40 mM) or acetaldehyde (5-40 microM). Very low ethanol concentrations (2.5 mM) reduced testosterone production significantly, whereas even 40 microM acetaldehyde added repeatedly to the incubation medium had no effect on testosterone levels. An inhibitor of alcohol dehydrogenase, 4-methylpyrazole, abolished the ethanol-induced inhibition of testosterone synthesis. A suggested mechanism for the inhibition is an elevated free [NADH]/[NAD+] ratio in Leydig cells caused by the metabolism of ethanol.