Ethanol was found to suppress gonadotropin-stimulated testosterone biosynthesis in sexually mature male rats in vivo and in intact Leydig cells in vitro. A similar ethanol dose-response inhibition curve was also observed when either whole or homogenized cells were stimulated by dibutyryl cyclic AMP. In the presence of pharmacologically relevant ethanol concentrations, added NAD+ restored testosterone production to stimulated control levels. These results demonstrate a direct inhibitory effect of ethanol on testicular testosterone synthesis. The site of inhibition is primarily intracellular and the mechanism is probably through a decrease in the NAD+/NADH ratio caused by ethanol oxidation.