This study was designed to examine the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid production in human luteinizing granulosa cells (hLGC). TCDD (10 nM) or its solvent was added at the time of changing medium, directly to the cells, every 48 h for 8 days. To test the hypothesis that TCDD reduces estradiol (E₂) synthesis by an effect on cytochrome P450 aromatase, aromatase protein and aromatase activity were evaluated. E₂ decreased without changing either aromatase protein or its enzyme activity, suggesting that the target of toxicity of TCDD is upstream of aromatase in the steroidogenic pathway. When hLGC were incubated in the presence of labeled E₂, no changes in the metabolism of E₂ by treatment were observed. Since TCDD did not change progesterone or 17α-hydroxyprogesterone, the inhibition of E₂ synthesis by TCDD would seem not to involve steps such as cholesterol transport. Furthermore, the TCDD effect on E₂ concentration in these cells disappeared in the presence of excess androgens. We conclude that the inhibition of E₂ secretion by TCDD involves intermediate steps, specifically, the provision of androgens for aromatization.