The enzyme steroid 5-alpha reductase is responsible for the conversion of testosterone to dihydrotestosterone, the steroid that mediates the intracellular action of androgens in some target tissues. The goal of this study was to check the accuracy of three known biochemical methods of studying steroid 5-alpha reductase activity expressed by dermal fibroblasts, isolated from pubian skin. These methods were performed on cell lysates (spectrophotometric and spectrofluorimetric methods) and on cell culture media (Reversed Phase-HPLC) with the purpose of their use in diagnosis and monitoring of hyperandrogenic patients. We also optimized a molecular study of expression of 5-alpha reductase isoenzymes and used it in the analysis of patients diagnosed with polycystic ovary syndrome (PCOS) and hirsutism by comparison with normal women. There was noticed an increase of the isoenzyme expression level both in patients with PCOS and in the case of patients with hirsutism. In other experiments, dermal fibroblasts originating in 15 individuals were treated with androgen hormones (testosterone: 10(-7) - 10(-9) M) with the purpose of demonstrating the effect of hyperandrogenemia on the expression level of 5-alpha reductase isoenzymes. The study of 5-alpha reductase type 1 mRNA expression levels in fibroblasts resulted from 4 normal individuals, 3 patients with hirsutism and 6 patients with PCOS, demonstrated an increase with 108.3% at the patients with PCOS and 47.3% at the patients with hirsutism compared with normal women. We concluded that hyperandrogenemia is associated with high levels of expression of 5-alpha reductase type 1 and, to a less extent, of type 2 isoenzyme in pubian skin cultured fibroblasts.