Four cell lines, each derived from a primary tumor from a patient with breast carcinoma, were grown to confluence in alpha-Minimum Essential Medium with 15% fetal calf serum and incubated for 24 h with [3H]androstenedione. The two lines (SA and PP) with the lowest formation of estrone and estradiol (less than 0.1% conversion) were the most active in the formation of the 5 alpha-reduced androgen metabolites androsterone (AND), 5 alpha-androstanedione (5 alpha-A-dione), and dihydrotestosterone (DHT). The two lines with the highest aromatase activity (DM and MD) had the lowest formation of 5 alpha-reduced metabolites. To determine if the 5 alpha-reduced androgen metabolites formed within the breast carcinoma cells could influence aromatase activity, the MD line was further studied. After 24-h preincubation with AND, DHT, or 5 alpha-A-dione at concentrations of 10(-6), 10(-7), and 10(-8) M, [3H]androstenedione was added to the culture medium, and aliquots were removed at 0, 4, 8, and 24 h. An 8-h incubation period was found to be optimum for inhibition studies. In comparison to control levels of estrone (2.5%) and estradiol (0.35%) formation, inhibition of aromatization was evident with all three compounds at 10(-8) M, with 5 alpha-A-dione producing the greatest inhibition (50%). At 10(-7) M, inhibition ranged from 45% (AND) to 70% (5 alpha-A-dione), and at 10(-6) M, inhibition was greater than 90% for each compound. 5 alpha-A-dione produced slightly greater inhibition than AND or DHT at each concentration tested. Since each of these compounds was capable of inhibiting aromatization, the cumulative effect of these 5 alpha-reduced metabolites could be an important factor in the intracellular regulation of aromatase activity.