Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7/LCC1 and MCF-7/LCC2. Using the product isolation method, the activity of aromatase, 5alpha-reductase, 3alpha/beta-hydroxysteroid oxidoreductase and 17beta-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E3), estradiol (E2), estrone (E1), 3alpha/beta-androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [14C]T or [14C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [14C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7/LCC1 and MCF-7/LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [14C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7/LCC1 and MCF-7/LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.