Androgens are an integral part of human physiology. The de novo production of androgens is generally limited to the adrenal cortex and the gonads. Androgen synthesis by these steroidogenic tissues requires the bifunctional enzyme cytochrome P450c17, which catalyzes both 17 hydroxylase and 17,20 lyase activities. 17,20-lyase activity is relevant to the regulation of androgen production, and is allosterically modulated through the action of an accessory protein, cytochrome b5 (CytB5). Our objective was to determine the cellular localization of P450c17 and CytB5 in androgen-synthesizing tissues of the human. Immunohistochemical analyses of P450c17 and CytB5 were performed on fetal and adult human adrenals, ovaries, and testes. In the fetal adrenal, CytB5 and P450c17 were both found in the cells of the fetal zone, but not in the neocortex. In the adult adrenal, the zona fasciculata was immunoreactive for P450c17 only, whereas the zona reticularis was immunopositive for both P450c17 and CytB5. In the adult gonads, P450c17 and CytB5 were colocalized in the Leydig cells of the testis, theca interna cells of the follicle, theca lutein cells, and isolated cell clusters in the ovarian stroma. Whereas P450c17 and CytB5 were colocalized in the Leydig cells of the fetal testes, there was no immunostaining for either in the midgestational fetal ovary. Our findings of colocalization of CytB5 and P450c17 are strongly supportive of the view that CytB5 plays an important role in the regulation of the androgen biosynthetic pathway in the fetal and adult human.