AIMS: To characterize the subepithelial (SE) stromal cells in normal human prostate and to study phenotypical changes in these SE stromal cells in benign prostate hyperplasia (BPH) and prostate cancer (PCa). METHODS AND RESULTS: Tissue was obtained from normal, hyperplastic and tumoral areas in prostatectomy specimens. Tissue samples were processed for morphology, immunohistochemistry, immunofluorescence and electron microscopy. An ultrastructural and immunohistochemical phenotype for the SE stromal cells was assessed, and changes occurring in BPH and PCa were looked for. Based on the ultrastructural findings the dominant SE stromal cell types in normal prostate were interstitial Cajal-like cells and spindle-shaped myoid cells with both myoid and fibroblastic features. An immunohistochemical correlate was found with selective expression of mesenchymal and myoid cell markers. In BPH and PCa, a changed stromal cell composition of the SE region was found. Furthermore, direct contacts between spindle-shaped myoid cells and tumour cells were observed in PCa. CONCLUSIONS: The present study shows for the first time that the SE area in the human prostate houses different stromal cell types with distinct phenotypes. In BPH and PCa specific disease-related changes were observed in the organization and phenotypes of SE interstitial cells.