The effects of ethanol on the concentrations of steroids in testis was studied in adult rats. Testosterone, seven of its potential precursors, three of its metabolites, and estradiol were analyzed by gas chromatography-mass spectrometry of samples from testes removed 2 h after intraperitoneal administration of ethanol, 1.2 g/kg body weight. The same analyses were made on samples from control rats. Ethanol gave a marked increase of all 3 beta-hydroxy-delta 5 steroids analyzed: pregnenolone (60%), 17-hydroxypregnenolone (480%), dehydroepiandrosterone (430%) and 5-androstene-3 beta, 17 beta-diol (60%). This resulted in highly significant increases of the 3 beta-hydroxy-delta 5/3-oxo-delta 4 steroid ratios for all steroid couples analyzed. An analogous increase of the ratio between 5 alpha-androstane-3 beta, 17 beta-diol and dihydrotestosterone was also observed, whereas the ratio between androstenediol and dehydroepiandrosterone was decreased by ethanol. The concentration of estradiol was not affected. The results indicate that moderate doses of ethanol inhibit the conversion of 3 beta-hydroxy-delta 5 to 3-oxo-delta 4 steroids. This may be one mechanism by which ethanol decreases the production of testosterone.