Recent epidemiologic studies suggest that women smokers have lower endogenous estrogen levels than nonsmokers. The effects of aqueous extracts of cigarette smoke on aromatase were evaluated in cultures of human granulosa cells. Aqueous extracts of cigarette smoke inhibited the conversion of androstenedione (delta 4A) to estradiol in a dose-dependent manner. Dialysis experiments demonstrated that 90% of the inhibitory activity of aqueous extracts of cigarette smoke was in the less than 1000 mol wt fraction. Removal of the aqueous extract of cigarette smoke from the culture medium resulted in a complete reversal of the inhibition of delta 4A aromatization. Addition of supraphysiologic concentrations of delta 4A (73 microM) to the culture medium blocked the smoke-induced inhibition of aromatization. Two low-molecular-weight components of cigarette smoke, nicotine and anabasine, inhibited granulosa cell aromatase in a dose-dependent manner. These studies suggest that constituents of cigarette smoke inhibit a major steroidogenic pathway.