Human granulosa cells cultured in the presence of estradiol, FSH, and LH secreted relatively little progesterone during the first 3–5 days of culture. The cells then underwent a morphological transformation associated with a 10-fold or more increase in progestin production. Cells not cultured with the tropic hormones usually produced progestin at a lower rate. Serum was required for the expression of maximal steroidogenic activity. Inhibitors of de novo sterol synthesis, ML-236B and 5-cholesten-3β,25-diol, did not affect progestin secretion by luteinized cells cultured in 20% serum, but these inhibitors did reduce [14C]acetate incorporation into sterols by 90%. ML-236B also did not affect hCG-stimulated progesterone secretion by the cells.However, when the cells were changed into lipoprotein-deficient medium, progestin production was markedly reduced within 24 h. The addition of low density lipoproteins (LDL) to the culture fluid restored progestin production, whereas high density lipoproteins tended to further inhibit steroid secretion. The effects of LDL on steroidogenesis were dose dependent and saturable. We conclude that luteinized human granulosa cells use LDLcarried sterol as the primary substrate for steroidogenesis.