Objective: To evaluate adrenal and ovarian steroidogenesis before and after long-term treatment with metformin in women with polycystic ovary syndrome (PCOS). Design and Methods: Twenty-four women with PCOS were evaluated before and after treatment (27 +/- 4 weeks) with metformin (1000 mg/day) using adrenocorticotrophin (ACTH), GnRH analogue and oral glucose tolerance (oGTT) tests. For statistical evaluation, ANOVA and Wilcoxon's test were used. Results: In 58% of the women a significant improvement in menstrual cyclicity was observed. No significant change in basal steroid levels was found. After ACTH stimulation, a significant decrease in the activity of 3 beta -hydroxysteroid dehydrogenase in C-21 steroids (P < 0.05) and in 17-hydroxysteroid dehydrogenase (P < 0.01) was observed, as was an increase in the activity of C17.20-lyase in the (4) pathway (P < 0.01). A significant growth in the dehydroepiandrosterone (DHEA)/DHEA-sulfate ratio (P < 0.05) was detected. With regard to ovarian steroidogenesis, a significant decrease in the stimulated levels of testosterone (P < 0.05), index of free testosterone (P < 0.01), LH (P < 0.05) and oestradiol (P < 0.01), and an increase in the levels of 17-hydroxypregnenolone (P < 0.05) were detected. In the indices of ovarian enzyme activities, we observed a significant decrease in 3-hydroxysteroid dehydrogenase in C-21 steroids (P < 0.01), in C17.20-lyase in the 115 pathway (P < 0.01), in 17 beta -hydroxysteroid dehydrogenase (P < 0.05) and in aromatase. In glucose metabolism, a tendency towards reduction in the homeostasis model assessment (HOMA)-R (for insulin resistance) and HOMA-F (for cell function) was detected. In addition, an increase in the levels of C peptide during oGTT was observed (P < 0.01). Conclusions: Long-term metformin treatment reduced various steroid enzymatic activities both in the ovary and the adrenal glands, without apparent changes in basal steroid levels and in insulin sensitivity.