The cytosolic members of the heat shock protein 70 (hsp70) family have in recent years been shown to elicit protective immunity mediated via binding to antigenic peptides in tumor and viral animal models. In this study we have used the methylcholanthrene (MC)-induced sarcomas MC57S and MC57X, previously shown to express individually distinct MHC-I associated peptides recognized by tumor necrosis factor-alpha (TNF-alpha) producing CD8(+) T cells. With hsp70 purified from tumor or liver tissue, we were able to confirm that tumor-derived hsp70 elicited in vivo protection against a challenge with the same tumor as that used for hsp70 isolation. We also observed that immunizing with hsp70 isolated from tumor tissue resulted in a significantly better protection than immunizing with hsp70 isolated from the liver tissue of healthy mice. In two out of three experiments however, immunization with liver-derived hsp70 as well as tumor-derived hsp70 resulted in a significantly delayed tumor outgrowth as compared to saline-injected controls. In vitro, T cell lines from mice immunized with tumor-derived hsp70 could recognize tumor cells from the same MC57 tumor as that used for the hsp70 purification, resulting in TNF-alpha production. In sera from hsp70-immunized mice we observed undetectable or only very low levels of anti-hsp70 antibodies, suggesting that it is possible to repeatedly immunize the mice with no significant interference from neutralizing antibodies. We therefore conclude that hsp70 immunization can lead to protection against the tumor it was purified from, with a low risk of eliciting neutralizing antibodies that could affect further immunizations.