SUMMARY Human granulosa, thecal, and stromal cells were grown in tissue culture in a medium containing 15% pooled human male serum, 30% Parker's medium '199', and 55% Hanks's solution. Granulosa cells underwent hyperplasia until about 10–12 days of culture and stayed alive up to at least 100 days. However, from about 20 days onward some of the cells gradually became detached from the glass. Initially the cytoplasm became eosinophilic and granular and accumulated lipid droplets and maintained these characteristics for about 20 days. From day 0–3 of culture, cells from non-gonadotrophin-treated women secreted 10·7 μg. progesterone, 0·54 μg. pregnenolone, 0·52 μg. oestrone and 0·45 μg. oestradiol as determined by gas-liquid chromatography. They also converted pregnenolone to similar products. A pool of thecal cells cultured for 3 days secreted 2·84 μg. progesterone, 0·36 μg. pregnenolone, 1·38 μg. 17-hydroxyprogesterone, 2·10 μg. androstenedione, 1·93 μg. oestrone and 1·44 μg. oestradiol. Stromal cells secreted less steroid per cell and no detectable oestrogens. Thecal cells grew in stellate colonies and had a lacy cytoplasm which contained vacuoles of various sizes which did not contain large amounts of lipid. Stromal cells resembled fibroblasts in morphology and growth characteristics.