Altered peptide ligands (APLs) with enhanced binding to MHC class I can increase the CD8(+) T cell response to native Ags, including tumor Ags. In this study, we investigate the influence of peptide-MHC (pMHC) stability on recruitment of tumor Ag-specific CD8(+) T cells through cross-priming. Among the four known H-2(b)-restricted CD8(+) T cell determinants within SV40 large tumor Ag (TAg), the site V determinant ((489)QGINNLDNL(497)) forms relatively low-stability pMHC and is characteristically immunorecessive. Absence of detectable site V-specific CD8(+) T cells following immunization with wild-type TAg is due in part to inefficient cross-priming. We mutated nonanchor residues within the TAg site V determinant that increased pMHC stability but preserved recognition by both TCR-transgenic and polyclonal endogenous T cells. Using a novel approach to quantify the fraction of naive T cells triggered through cross-priming in vivo, we show that immunization with TAg variants expressing higher-stability determinants increased the fraction of site V-specific T cells cross-primed and effectively overcame the immunorecessive phenotype. In addition, using MHC class I tetramer-based enrichment, we demonstrate for the first time, to our knowledge, that endogenous site V-specific T cells are primed following wild-type TAg immunization despite their low initial frequency, but that the magnitude of T cell accumulation is enhanced following immunization with a site V variant TAg. Our results demonstrate that site V APLs cross-prime a higher fraction of available T cells, providing a potential mechanism for high-stability APLs to enhance immunogenicity and accumulation of T cells specific for the native determinant.