3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)