We have isolated UV light-induced and spontaneous tumors along with nonmalignant cells and tissues from each host. CD8+ CTL clones generated to a number of highly immunogenic UV-induced tumors did not react with autologous normal fibroblasts nor with autologous second tumors. Using up to 25 independently induced tumors as targets, these CTL clones were found to be uniquely specific for the particular tumor used for immunization even when multiple tumors isolated from the same animals were used as targets. In addition to this extensive antigenic diversity of independently induced tumors, we found that a single cancer cell can express multiple independent antigens that were uniquely expressed on the tumor but were not detectable on autologous nonmalignant fibroblasts. A poorly immunogenic spontaneous tumor was also found to express an antigen that was uniquely specific for the immunizing tumor in that it was absent from any of 25 other tumors tested. This antigen was recognized by a mAb and not detected on autologous nonmalignant fibroblasts or on an autologous second spontaneous tumor. These findings demonstrate that syngeneic CTL clones or mAbs can define unique antigens on UV-induced or spontaneous tumors. The use of autologous nonmalignant fibroblast targets made it unlikely that these antigens were widely expressed on normal cells. The availability of cloned immunological probes to antigens on tumors isolated with autologous normal cells will allow a reliable identification of the genetic origins of unique antigens on experimentally induced and spontaneous tumors and permit a decisive answer to whether these unique antigens are encoded by normal genes or by genes that have undergone somatic mutations; i.e., whether these antigens are truly tumor specific.