Chronic administration of the estrogen 17 beta-estradiol induces kidney tumors in male Syrian hamsters within 6 months of initial exposure. Although these tumors have previously been studied histologically and histochemically and have been postulated to be derived from proximal tubular and/or interstitial cells, there exists no unambiguous evidence for an epithelial or mesenchymal origin. To elucidate the histogenesis of these neoplasms, kidney sections of hamsters treated with estradiol for 4, 5, and 6 months and age-matched untreated controls were investigated histologically and histochemically. Proliferating foci were observed in kidneys exposed to estradiol for 5 and 6 months. They consisted of clusters of spindle-shaped cells forming solid blocks, cords, or branches located between tubules. These foci were judged to be precursors of larger tumors identified in the latter treatment group. The histological and histochemical profile of foci and tumors matched closely. These lesions were marked by very high activities of alkaline phosphatase, adenyl cyclase, and glucose 6-phosphate dehydrogenase. In contrast, glycogen content and activities of glucose 6-phosphatase, succinate dehydrogenase, and gamma-glutamyl transpeptidase were low or absent. Immunofluorescence of the intermediate filaments revealed that foci and tumors solely expressed vimentin and desmin but not cytokeratin. The morphology, enzyme histochemical pattern, and immunofluorescence strongly support a mesenchymal origin of the estradiol-induced hamster kidney tumors studied. The neoplasms were probably derived from vascular smooth muscle cells of a cell subtype particularly sensitive to hormonal stimulation and transformation.