Although it is well established that ethanol suppresses gonadotropin- and cAMP-stimulated testicular steroidogenesis, there is not good agreement on two issues: which is the step in testosterone's biosynthetic pathway affected by ethanol; and the role of alterations in the NAD+/NADH ratio in ethanol's effects. In these studies, we have identified major differences between in vivo and in vitro approaches, which have previously been considered as totally equivalent experimental paradigms, which could explain these discrepancies. Under in vitro conditions, we observed that ethanol selectively inhibited the conversion of androstenedione to testosterone, but that it had a much more general effect under in vivo conditions. In addition, in agreement with other studies, NAD+ overcame ethanol's effects on testicular steroidogenesis in vitro, but only when labeled or unlabeled pregnenolone was added. In the absence of added pregnenolone, NAD+ was not effective in preventing ethanol's effects. Our results, thus, indicate that the differences which currently exist in the literature may be explained by the indiscriminate usage of in vivo and in vitro techniques.