Naturally processed peptides, obtained by acid extraction of tumor cells, contain Ags able to activate specific CTL in vitro. We recently reported that the nonprofessional APC, RMA-S, expressing the B7.1 molecule (RMA-S/B7), pulsed with naturally processed peptides from the nonimmunogenic B16F1 melanoma (B16F1a.e.) primed syngenic CD8+ T cells against the tumor in vitro. Here, we show the rejection of B16F1 melanoma by C57BL/6 mice after immunization with RMA-S/B7 cells pulsed with B16F1a.e. This response is critically dependent on both CD4+ and CD8+ cells, but not on NK cells. However, only CD8+ T cells exert anti-B16F1 cytolitic activity in vitro. Moreover, RMA-S/B7 cells pulsed with B16F1a.e. can be used to prevent the growth of 24-h preestablished melanomas. These results may have important implications for the clinical use of natural peptide fractions of tumor cells as therapeutic cancer vaccines.