The presence of tumor-specific or tumor-associated antigens in primary urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in ACI/N rats was examined by means of mixed lymphocyte-tumor cell culture. Bladder tumors (papilloma with cancer foci or cancer) induced by BBN treatment for 18 weeks or more (BT), epithelial cells from papillary hyperplasia induced by 6-week BBN treatment (PH), and normal bladder epithelial cells (NBE) were isolated from the bladder and used as stimulator cells after mitomycin C treatment. BT, PH and NBE all strongly stimulated the blastogenesis of the autologous lymphocytes on culture in medium containing fetal calf serum (FCS), but the spleen cells also stimulated the autologous lymphocytes in this medium. Although the magnitudes of blastogenesis were lower than those in FCS medium, on culture in medium containing rat serum, BT, PH and NBE, but not spleen cells, stimulated the blastogenesis of the autologous lymphocytes. The response with BT was significantly higher than the response with NBE, but there was no statistically significant difference between the response with BT and the response with PH. These results suggest that FCS medium, but not medium with rat serum, evokes the blastogenic response of lymphocytes cultured with autologous spleen cells, and that blastogenic responses with autologous bladder cancer can be produced by stimulation with some sort of tissue antigen present in normal or pre-neoplastic urinary bladder epithelial cells.