The in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid metabolism in human luteinized granulosa cells (hLGC) have been summarized as a decreased estradiol (E₂) production without altering either E₂ metabolism or cytochrome P450 aromatase activity. In the present study, hLGC were used to analyze the fate of different substrates for cytochrome P450 17α-hydroxylase/17,20-lyase (P450_c17) in the presence or absence of TCDD. Human LGCs were plated directly on plastic culture dishes in medium supplemented with 2 IU/ml of hCG. TCDD (10 nM) or its solvent was added directly to the cells at the time of medium change, every 48 h for 8 days. The objective of the experiment was to test the hypothesis that exogenous steroid, substrate for P450_c17, would reduce the TCDD effects on E₂ synthesis. With dehydroepiandrosterone (DHEA) (a P450_c17 product), a dose-related increase in E₂ production was observed and the effect of TCDD on lowering E₂ production disappeared. In contrast, with increasing doses, up to 10 μM, of pregnenolone (P₅), no change in E₂ production was observed. However, 17α-hydroxypregnenolone (17P₅) at 10 μM produced a modest but significant increase in the E₂ production. Treatments with P₅ and 17P₅ did not alter the effect of TCDD on E₂ production. Radiolabeled substrate utilization by hLGC suggests that the principal metabolic pathway for Δ5 substrates is the conversion to a Δ4 product probably by a very active 3β-hydroxysteroid dehydrogenase. We conclude that estrogen production by hLGC is limited at the level of lyase activity. Thus, these data suggest that the most likely target for the TCDD-induced inhibition of estrogen synthesis by hLGC is the 17,20-lyase activity of the P450_c17 enzyme complex.