Continuous culturing of two distinct human prostate specimens in the presence of interstitial collagenase added directly to conventional medium resulted in the isolation and extended growth of primary epithelial prostate cell (PEPC) cultures from each. Both continued to proliferate substantially beyond the average time determined for analogous untreated epithelial prostate isolates. Both repeatedly stain positive for keratin and are characteristically epithelial in morphological appearance and growth model. Both express androgen receptor mRNA and stain positive for androgen receptors. PEPC-2 displays an androgen dose-dependent stimulation of cell proliferation, as well as specifically binding 3H-R1881. PEPC-1 exhibits a hypotetraploid karyotype with loss of the Y chromosome. PEPC-2 conserves a normal human ploidy, including the Y chromosome, although there is extensive random chromosome loss. Elimination of the collagenase from the medium resulted in decreased cellular proliferation and accumulation of stainable collagen in both PEPC cultures. Neither PEPC isolate produced tumors in male nude mice, whether injected alone, mixed with matrigel, or combined with prostate or bone fibroblastic cells.