A novel human prostatic stromal cell culture, designated DuK50, has been passed in vitro > 12 mo. Tissue cultures were obtained from material harvested within a normal region of a radical prostatectomy specimen. These monolayers exhibited normal fibroblastic characteristics with each cell having a flattened, elongated appearance. Karyotypic analysis revealed a normal, male 46, XY chromosomal content with no numerical or structural abnormalities. DNA analysis using a Cell Analysis Systems Image Analyzer confirmed a euploid DNA content (7.9 pg DNA). Cellular markers for verification of stromal cell type were performed by immunohistochemical techniques. DuK50 stained positive for vimentin and fibronectin. Immunostains for epithelial cytokeratins and prostate-specific antigen were negative, which ruled out contamination with prostatic epithelial cells. Negative immunostaining with desmin monoclonal antibody and light staining with smooth muscle actin alpha is consistent with the staining pattern of myofibroblasts. Response to various androgens, measured by a microculture tetrazolium assay technique, revealed a significant growth stimulation of DuK50. Soft agar invasiveness assays and tumorigenicity studies in nude mice were negative. DuK50 exhibits a rapid doubling time with excellent plating efficiency, thrives in a readily available media supplemented with fetal bovine serum, and passes with routine trypsin protocols. The availability of this prostatic stromal cell culture may facilitate studies on this cell type's role in growth factor modulation, drug and steroid metabolism, and stromal-epithelial interactions in the prostate.