The enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta-HSD/I) is an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3 beta-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3 beta-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3 beta-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3 beta-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3 beta-HSD/I associated with the mitochondrial fraction did not require addition of exogenous NAD+. When the pyridine nucleotide was reduced following addition of substrates of the tricarboxylic acids cycle, the mitochondrial 3 beta-HSD/I activity decreased, suggesting that the enzyme utilizes NAD+ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD+. Submitochondrial fractionation disclosed that 3 beta-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3 beta-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a special organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3 beta-HSD/I activity.