A study was made of the auto-proliferative activity of human renal-carcinoma cells in the supernatant from a carcinoma-cell culture in serum-free medium to which an anticancer agent had been added (5-FU). The human renal-cancer cells used in this study were of 3 strains: ACHN, VMRC-RCW and NT. When each line was cultured in medium containing no 5-FU, the supernatant showed almost no activity for stimulating DNA synthesis. However, when the line was cultured in the presence of 5-FU, the supernatant showed autocrine growth-promoting activity which strongly stimulated DNA synthesis of 3 renalcancer cell lines in a dose-dependent manner. Activity could be detected in a 4to 6-kDa fraction by gel filtration. This fraction increased the DNA-synthesis-promoting activity of epidermal growth factor, transforming growth factor+, basic fibroblast growth factor and insulin, and was acid-and heat-stable. It was also stable against pepsin and dithiothreitol. DNA synthesis in BALB/c 3T3. adult rat hepatocytes and rabbit renal tubular cells was not affected by this fraction which was thus considered not to affect non-cancerous cells. Renal-cell carcinoma responds poorly to anticancer agents, and the autocrine activity of the fraction may possibly be a factor accounting for this resistance.