Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.