The aim of this study on testosterone (T) metabolism in benign prostatic hyperplasia (BPH) and prostatic cancer was to compare the formation of metabolites in freshly isolated epithelial cells and in cells of long-term cultures (2 passages) and to identify the 5alpha-reductase (5alpha-R) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms responsible for metabolite formation. Androst-4-enedione (A), dihydrotestosterone (DHT) and 5alpha-androstanedione (5alpha-A) formation were measured by high-performance liquid chromatography coupled to a Flo-one HP radioactivity detector. Enzyme isoforms were studied by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). T conversion into A by 17beta-HSD, rather than reduction into DHT by 5alpha-R, was by far the predominant activity in cultured epithelial cells. The metabolic profile did not differ substantially between BPH and cancer cells. Long-term cell culture led to an increase in A formation compared with the level recorded in freshly isolated cells, with no significant incidence on the relative DHT level. According to RT-PCR results, both 5alpha-R isoforms (1 and 2) and 2 17beta-HSD isoforms (2 and 3) are present in epithelial cell cultures and in tissues. According to Northern blot analyses, the mRNAs for 5alpha-R2 and 17beta-HSD4 are expressed in tissue and those for 5alpha-R1 and types 2 and 4 17beta-HSD in isolated cell cultures. Moreover, finasteride, a specific 5alpha-R2 inhibitor, inhibits DHT and 5alpha-A formation in long-term cell culture of adenocarcinoma epithelial cells plated on Matrigel, suggesting a 5alpha-R2 expression. Thus, although 5alpha-R2 is present in freshly isolated epithelial cell cultures and in long-term epithelial cells cultured on Matrigel and predominates in prostate tissue, it is the 5alpha-R1 isoform that is preferentially expressed in epithelial cell cultures.