Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5alpha-dihydrotestosterone are produced from testosterone by aromatase and 5alpha-reductase, respectively. Metabolites of vitamin D have shown to inhibit the growth of prostate cancer cells. The aim of the present study was to verify whether 25-hydroxyvitamin D(3) (25OHD(3)), 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)], dexamethasone, and progesterone regulate the expression of aromatase and 5alpha-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD(3), 1alpha,25-(OH)(2)D(3), dexamethasone, or progesterone. Aromatase and 5alpha-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [(3)H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10nM dexamethasone, 1-100 nM 1alpha,25-(OH)(2)D(3) and 100 nM-10 microM progesterone. The induction was enhanced when hormones were used synergistically. Real-time RT-PCR analysis showed no regulation of the expression of aromatase mRNA by any steroids tested in either LNCaP or PC3 cells. The expression of 5alpha-reductase type I mRNA was not regulated by 1alpha,25-(OH)(2)D(3) and no expression of 5alpha-reductase type II was detected in LNCaP.