BACKGROUND. An in vitro model of prostatic stromal cells suitable for experimental studies of the pathogenesis of BPH is still lacking. We therefore standardized the isolation, cultivation, and characterization of human prostatic stromal cell lineages. METHODS. Stromal cells were isolated from a surgical specimen of BPH. Using antibodies specific for either epithelial or stromal cells of the human prostate, the isolated cells were morphologically and immunohistochemically characterized. Viability and functional activity were assessed by proliferation assays and stimulation experiments. Gene expression was monitored by RT-PCR. RESULTS. In early passages (P8), cells showed a high purity (Ն98%) for stromal markers; about 60% displayed the characteristics of fibroblasts, and the remaining 40% were classified as smooth muscle cells. In late passages (P20), the proportion of muscle cells declined to 10%. Stimulation experiments including basic fibroblast growth factor (bFGF) resulted in enhanced proliferation, whereas dihydrotestosterone (DHT), estrogen, and flutamide did not influence proliferation. Gene expression studies demonstrated a positive signal for androgen receptor and keratinocyte growth factor (KGF). CONCLUSIONS. Prostatic stromal cells can be propagated several times and show karyotypic stability for up to 18 subculture experiments. The ratio of myoid and fibroblastic cells can be used for standardization of cell cultures with stable characteristics. Prostate 43: 20-30, 2000.