A modified radioassay for the determination of the 3 beta-hydroxy-delta 5-steroid dehydrogenase (3-beta-HSD) is described. The assay is based on the conversion of [3H]pregnenolone to [3H]progesterone followed by a digitonin precipitation step. The method was applied to neurons, glial cells, C6 glioma cells and adrenal tumor cells in culture. Adrenal tumor cells and C6 glioma cells showed higher enzyme activity than primary cultures of astrocytes and neurons. Dependence of enzyme activity on pH, protein concentration and reaction time was demonstrated for C6 cells. A pH optimum was shown between 7.5 and 8.1, and the reaction was linear up to 2 h. beta-oestradiol inhibited 3-beta-HSD activity completely. The assay presented is fast, highly reproducible, and offers the possibility of studying 3-beta-HSD activity in differentiating cells in culture without preparation of microsomes or extraction of reaction products.