An immortalized human prostate stromal cell line (PS30) was previously established using recombinant retrovirus encoding human papillomavirus 16 gene products. In this study, we further characterize this stromal cell line for its potential use in a stromal–epithelial coculture model for prostate cancer prevention. Using reverse transcriptase–polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry, we examined expression of androgen receptor (AR), vitamin D receptor (VDR), prostate-specific antigen (PSA), transforming growth factor-β (TGF-β), and insulin-like growth factors (IGF) families and their receptors, metalloproteinases (MMP) MMP-2 and MMP-9, as well as the cells' ability to respond to the synthetic androgen R1881. The PS30 stromal cells do not express PSA, confirming their stromal origin. They are positive for both AR messenger ribonucleic acid (mRNA) and protein; however, they do not respond to growth stimulation by the synthetic androgen R1881. The PS30 cells express mRNA for VDR, TGF-βs, IGFs and their receptors, as well as the MMPs. Moreover, they produce significant amounts of TGF-β1, TGF-β2, IGFBP-3, and MMP-2 proteins. Our observations confirm the use of PS30 for the study of stromal–epithelial interactions in the modulation of prostate carcinogenesis.