Delayed-type hypersensitivity (DTH) responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum-) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum- antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum- peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8+ cell-mediated DTH to P815AB was unaffected by the ablation of CD4+ cells, the same treatment, or neutralization of IFN-gamma, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4+ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum- cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.