The expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in steroidogenic tissues is an absolute requirement for mammalian reproduction, fetal growth, and life maintenance. We sought to identify extraglandular tissue sites in the human fetus where 3 beta HSD is expressed. To this effect, we conducted in vitro studies by use of homogenates prepared from second trimester fetal tissues. To facilitate the determination of 3 beta HSD activity, an abbreviated technique was developed that consisted in the use of [3 alpha-3H]dehydroepiandrosterone [( 3 alpha-3H]DHEA) as the substrate and NAD+ as the cofactor. With these reagents, the enzymatic reaction leads to the production of both nonradiolabeled androstenedione and NAD3H in equimolar amounts, and the radioactivity associated with NAD3H is used for quantification of 3 beta HSD activity. The kinetic isotope effect introduced by substitution of tritium for hydrogen at the C-3 alpha position of DHEA, determined with six different tissues, was 2.5 +/- 0.7 (mean +/- SD). The specific activities of the enzyme in peripheral tissues and ovary were relatively low, in the range of 0.03 nmol/mg protein.h for stomach (n = 2) to 0.18 +/- 0.14 nmol/mg protein.h for liver (mean +/- SD; n = 13), while in fetal testis and placenta the specific activities were relatively high, viz. 3.4 +/- 0.7 nmol/mg protein.h (mean +/- SD; n = 4) and 2.8 +/- 1.8 nmol/mg protein.h (mean +/- SD; n = 13), respectively. The findings of this study serve to demonstrate that 3 beta HSD is distributed widely among tissues of the human fetus. Although the enzymatic activity was easily demonstrated in peripheral tissues by the use of radiolabeled DHEA as the substrate, 3 beta HSD protein was not readily detected by Western analysis.