Three-beta-hydroxysteroid dehydrogenase (HSDB3) is the enzyme which catalyses the oxidative conversion of delta 5-3 beta-hydroxy steroids to the delta 4-3-keto configuration and is therefore involved in the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Deficiency of the enzyme is associated with congenital adrenal hyperplasia and is usually lethal in early life. Despite its crucial role, chromosome assignment of the gene for this enzyme has not been reported. Using in situ hybridization, we report that hybridization with labeled human HSDB3-specific cDNA yielded 27% of silver grains associated with chromosome 1 with a maximal concentration in the p13 band.