The metabolism of radiolabeled progesterone and androstenedione was evaluated in endothelial cells from human umbilical cord vein and arteries maintained in culture. The predominant metabolite of progesterone was 5α-pregnane-3,20-dione and that of androstenedione was 5α-androstane-3,17-dione. Thus, the major pathway of progesterone and androstenedione metabolism within these cells is via steroid 5α-reductase. The rate of formation of 5α-pregnane-3,20-dione from progesterone by venous endothelial cells was linear with incubation time up to 4 h and with cell number up to 1.6 × 10 6 cells/ml. The apparent K m of 5α-reductase for progesterone was 0.4 μM; and, the V max was 55 pmol 5α-pregnane-3,20-dione formed/mg proteinβ h. The rate of 5α-androstane-3,17-dione formation from androstenedione also was linear with incubation time up to 4h. in addition to 5α-androstane-3, 17-dione, the metabolism of androstenedione by either venous or arterial cells resulted in the formation of various minor metabolites, inclucling testosterone and 5α-reduced steroids, viz. 5α-dihydrotestosterone, androsterone, isoandrosterone, 5α-androstane-3α,17β-diol, and 5α-androstane-3β,17β-diol. Estrogens (i.e. estradiol-17β and estrone) were not detected as products of androstenedione metabolism. The formation of these metabolites are indicative that the steroid-metabolizing enzymes present in endothelial cells are: 5α-reductase, 17β-hydroxysteroid oxidoreductase, 3α-hydroxysteroid oxidoreductase, and 3β-hydroxy steroid oxidoreductase.