The present study was conducted to isolate and to characterize stromal cells from the human prostate and to study the effects of androgen and different growth factors in this model system. Benign prostatic hyperplasia (BPH) tissue samples were obtained from transurethral resection of the prostate (TURP). Tissue specimens were mechanically and enzymatically dissociated by treatment with DNAse and collagenase. Epithelial cells were separated from stromal cells by discontinuous Percoll gradient centrifugation. The stromal cells obtained were cultured in phenol red‐free RPMI‐1640 supplemented with 10% fetal bovine serum. Immunocytochemical analysis revealed that the stromal cell cultures were composed of both smooth muscle cells and fibroblasts. The short and broad, smooth muscle cells were identified by using an antibody directed against α‐smooth muscle actin. The thin and elongated fibroblasts stained positively for prolyl 4‐hydroxylase. Smooth muscle cells were the predominant cell type in the present investigation. Typical cultures contained up to 99% of cells staining positively for α‐smooth muscle actin. The prostate smooth muscle cultures were treated with dihydrotestosterone (DHT), bovine pituitary extract (BPE), basic fibroblast growth factor (bFGF) and transforming growth factor‐β (TGF‐β). When cells were cultured in serum free RPMI‐1640 supplemented with ITS+ (insulin, transferrin, and selenious acid) no significant (P > 0.05) stimulative effect after treatment with DHT or TGF‐β was detectable. Basic FGF had a slight but significant (P < 0.05) mitogenic effect in medium supplemented with ITS+. In the presence of 10% charcoal‐stripped fetal bovine serum (cFBS) DHT, at a concentration of 0.1 nM, was able to cause a slight but significant (P < 0.05) mitogenic effect on BPH smooth muscle cells growth. Basic FGF was able to stimulate BPH smooth muscle cells in a concentration‐dependent fashion. The combination of DHT and 0.1 ng/ml bFGF was able to increase the proliferation of prostate smooth muscle cells above either agents alone. Addition of BPE to serum free RPMI‐1640 caused a significant (P < 0.05) stimulation of cell proliferation in a concentration‐dependent fashion. Addition of TGF‐β to serum or BPE containing RPMI‐1640 caused a significant (P < 0.05) inhibition of cell proliferation in a concentration‐dependent fashion. TGF‐β was cytostatic to the benign prostatic smooth muscle cells only in the presence of media containing growth stimulating factors found in charcoal‐stripped serum or in bovine pituitary extract. These results demonstrated that stromal fraction isolated from BPH specimens was composed of both fibroblasts and smooth muscle cells. These cells could be cultured and were able to respond to various growth stimulatory and inhibitory agents. © 1996 Wiley‐Liss, Inc.