Kinetic constants for the 5 alpha-reductase were determined in freshly isolated epithelial cells from the rat ventral prostate. Studies were also performed on stromal tissue but not isolated stromal cells for comparison. Secretory and non-secretory epithelial cells were separated by centrifugation in a Percoll gradient. Both epithelial cell populations metabolized testosterone to predominantly 5 alpha-dihydrotestosterone (5 alpha-DHT), although when expressed per cell the capacity for conversion was 3-4-fold higher for secretory cells (7.4 pmol/min/10(6) cells) than for non-secretory cells (2.3 pmol/min/10(6) cells; P less than 0.01 in 4 separate studies). When compared per mg cytosol protein this difference became non-significant. Stromal tissue contained a 5 alpha-reductase Vmax (expressed) per mg protein) which was comparable to the non-secretory cell enzyme. Lineweaver-Burke plots revealed different Km values for the different cell populations (12.5, 5.9 and 4.7 microM for secretory, non-secretory and stromal cells, respectively) suggesting the presence of different isoforms of the enzyme, or differences in the intracellular concentrations of enzyme antagonists.