The normal developmental pattern of 17β-hydroxysteroid dehydrogenase (17HSD) activity in genital skin was examined using radiolabeled androstenedione as a substrate in a microassay based on high-pressure liquid chromatography separation of the metabolites. This assay allowed the simultaneous determination of 17HSD and 5α-reductase (5R) activities in both individual foreskin samples and pools of tissue obtained at circumcision from birth to 8 years of age. The results show that 17HSD activity is very low at birth and increases steadily during the so-called quiescent period. Reciprocal changes were observed for 5R. The increase in 17HSD activity appears to be independent of gonadal stimulation, but the mechanisms involved remain to be elucidated. From a clinical standpoint, our results provide an alternative explanation for the relative lack of virilization observed in newborns with testicular 17HSD deficiency.