Stromal cells are key regulators of growth and differentiation in the adult human prostate. Alterations in the stroma are believed to initiate the development of benign prostatic hyperplasia, and stromal-epithelial interactions may have a role in malignant progression. The prostatic stroma is composed of two major cell types, smooth muscle cells and fibroblasts. Cell cultures from the prostatic stroma have been established by several investigators, but the phenotype of these cells has not been extensively characterized and it is not clear whether they are fibroblastic or smooth muscle-like. In this study, the response of stromal cells cultured from normal prostatic tissues to transforming growth factor-beta (TGF beta) was investigated. We confirmed a previous report that TGF beta inhibited the growth of prostatic stromal cells in serum-containing medium, and showed that inhibition also occurred in serum-free medium. Growth inhibition by TGF beta was irreversible after 24 to 72 h of exposure. In the absence of TGF beta, cells were fibroblastic and expressed vimentin and fibronectin but little alpha-smooth muscle actin. After 3 days of exposure to 1 ng/ml of TGF beta, the majority of cells expressed alpha-smooth muscle actin and desmin, as demonstrated by immunocytochemistry and immunoblot analysis. This effect was specific and alpha-smooth muscle actin was not induced by two other growth-inhibitory factors, retinoic acid or 1,25-dihydroxyvitamin D3. These results suggest that TGF beta is an important regulator of growth and differentiation of prostatic stromal cells and that a smooth muscle cell phenotype is promoted in the presence of TGF beta.