Stromal growth is critical for prostate enlargement during benign prostatic hyperplasia (BPH). While responses of prostate cells to single growth factors have been well characterized, responses to multiple growth factors at once are poorly understood. Here, we examined the effects of combinations between epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor-β1 (TGF-β1) in human prostate stromal cells.EGF, FGF, and TGF-β1 were applied to WPMY-1 cells, an immortalized, non-malignant line of stromal cells from the human prostate. Hypertrophic responses were assessed by protein/DNA ratio, and cyclin D1 mRNA by RT-PCR. Expression of EGF, FGF, and TGF-β1 and their receptors in human prostate tissue was analyzed by RT-PCR, Western blot, and fluorescence staining.Hypertrophic responses to single growth factors and combinations were similar. Combinations showed additive effects on cyclin D1 mRNA. Combination of EGF with TGF-β1, but not EGF or TGF-β1 alone, caused assembly of cells to a new two-dimensional structure, being characterized by dense aggregates connected by branches of few cells. EGF and TGF-β1 were detected together in human prostates. Receptors for EGF and TGF-β colocalized on stromal cells in human prostates.Responses of prostate stromal cells to combinations of EGF, FGF, and TGF-β1 may be quantitatively different, qualitatively different, or similar to responses to single growth factors. The combination of EGF and TGF-β1, but not EGF or TGF-β1 alone, induces aggregation of prostate stromal cells, which may be relevant for morphogenesis.