The enzymatic activity of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD/I) constitutes an essential step in the biosynthesis of active steroid hormones such as progesterone, mineralo- and gluco-corticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal; however, 3 beta HSD/I activity is also present in mitochondrial preparations. In the present study, the distribution of 3 beta HSD/I in bovine adrenocortical subcellular preparations has been reexamined, and the catalytic properties of the enzyme present in the various cell compartments have been characterized. About 30% of the total 3 beta HSD/I was found to remain tightly associated with highly purified mitochondrial preparations. The preferred substrate of the mitochondrial enzyme was pregnenolone. Examination of submitochondrial preparations revealed that 3 beta HSD/I was associated with both the inner membrane and a particulate fraction that sediments in a density gradient between inner and outer membranes. The specific activity of the enzyme was at its highest in this intermediate density fraction, which exhibited the properties of mitochondrial intermembrane contact sites. Taken together, these observations suggest that these contact sites may represent a supramolecular organization of biological significance in adrenocortical cell steroidogenic functions. Such intermembrane fusion sites would facilitate the access of cholesterol to the inner membrane in which cholesterol side-chain cleavage cytochrome P-450 is located as well as the rapid transformation of its reaction product (i.e. pregnenolone) to progesterone by 3 beta HSD/I. Such a submitochondrial organization opens new possibilities in the understanding of the regulation of adrenocortical differentiated functions.