The role of mesenchymal-epithelial interactions in androgen action was explored using Sertoli cells as the epithelial cells and testicular peritubular cells or prostatic stromal cells as mesenchymal cells. Footsole fibroblasts served as a control. The secretion of transferrin was used as an androgen-regulated parameter of Sertoli cell function. It is demonstrated that coculture of peritubular or stromal cells with Sertoli cells markedly increases the production of transferrin. This effect requires a 4-day latent period and is maximal with low concentrations (10%) of mesenchymal cells. Stimulatory effects of androgens can only be demonstrated at suboptimal concentrations of the latter cells. Fibroblasts are inactive. At least two mechanisms contribute to these stimulatory effects. Peritubular cells and stromal cells share the ability to promote the deposition of an extracellular matrix when cocultured with Sertoli cells. When Sertoli cells are seeded on this matrix, the production of transferrin is increased. This effect requires no latent period and is independent of the presence of androgens during the period of matrix deposition. In addition, peritubular cells and stromal cells produce diffusible mediators which increase transferrin production by Sertoli cells. In both cell types, the production of these mediators is controlled by androgens, and their action is preceded by a 4-day latency period. The mediators have a comparable mol wt (45,000) and resemble P Mod-S, known to be secreted by peritubular cells. These data suggest that mesenchymal-epithelial interactions play a role in androgen-supported maintenance of adult function and that mesenchymal tissue from different androgen target tissues produces similar or identical mediators of androgen action.