One to five percent of human renal cell carcinomas are associated with polycythemia. It is generally assumed that polycythemia results from the secretion of erythropoietin (Epo) by the malignant cells. However, there is no direct proof supporting this hypothesis. Three patients with typical renal adenocarcinoma and polycythemia were studied. All three exhibited high Epo serum levels as measured by radioimmunoassay (RIA). A strong Epo signal was observed on Northern blot analysis of total RNA extracted from the renal tumors. The Epo message seemed to be of normal size and no Epo gene rearrangement was observed with the restriction enzymes tested. Using the in situ hybridization technique, a significant labeling was constantly observed on the tumor cells. Immunohistochemical studies showed that these tumor cells, known to be of tubular origin, were labeled by an anti-cytokeratin antibody and therefore were of epithelial nature. Thus, this study demonstrated that malignant cells of tubular origin were able to produce Epo constitutively, whereas in the mouse hypoxic kidney, peritubular cells (probably capillary endothelial cells) were the major site of Epo synthesis.