Primary cultured adult rat urothelial (RU) cells caused increased thymidine incorporation in rat bladder stromal (RS) cells in a coculture system. The concentrated conditioned medium derived from RU cell culture (CM-RU) also stimulated the growth of RS cells, and induced anchorage independent growth of NRK-49F cells. Since these activities were heat and acid resistant, we investigated whether epidermal growth factor (EGF) and/or transforming growth factors were the humoral factor(s) responsible. The immunodiffusion analysis of CM-RU gave a positive precipitin line with rabbit anti-rat EGF IgG but not with rabbit anti-rat transforming growth factor-alpha antibodies. The anti-rat EGF IgG inhibited CM-RU-stimulated thymidine incorporation into RS cells, whereas normal rabbit IgG did not. By immunofluorescent technique using rabbit anti-rat EGF antibodies, immunoreactive EGF was demonstrated in RU cells and the urothelial of cryoinjury-induced reparative hyperplasia. Immunofluorescent technique also demonstrated the presence of EGF receptors on the cell membrane of RU and RS cells, basal cells of normal rat urothelium, and cells of whole epithelial layers of reparative hyperplasia. These data strongly suggest that EGF or an EGF-like substance produced by RU cells and released into medium stimulates the growth of RS cells possibly mediated by EGF receptors.