We have demonstrated that in vivo administration of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to type I insulin-like growth factor receptor (IGF-IR) mRNA resulted in inhibition of C4HD breast cancer growth in BALB/c mice. The present study focused on whether in vivo administration of C4HD tumor cells pretreated with IGF-IR AS[S]ODN and irradiated could provide protection against C4HD wild-type tumor challenge and also on elucidating the mechanism mediating this effect. Our results showed that mice immunized with IGF-IR AS[S]ODN-treated C4HD cells experienced a growth inhibition of 53.4%, 61.6%, and 60.2% when compared with PBS-treated mice, wild-type C4HD cell-injected mice, or phosphorothioate sense oligodeoxynucleotide-treated C4HD cell-injected mice, respectively. The protective effect was C4HD-specific, because no cross-protection was observed against other syngeneic mammary tumor lines. The lack of protection against tumor formation in nude mice indicated that T cells were involved in the antitumoral response. Furthermore, cytotoxicity and splenocyte proliferation assays demonstrated that a cellular CD8(+)-dependent immune response, acting through the Fas/Fas ligand death pathway, could be mediating the antitumor effect induced by immunization with AS[S]ODN-treated cells. Immunization also induced splenocytes to produce Ag-dependent IFN-gamma, indicating the presence of a type 1 response. We demonstrated for the first time that IGF-IR AS[S]ODN treatment of breast cancer cells induced expression of CD86 and heat shock protein 70 molecules, both involved in the induction of the immunogenic phenotype. Immunization with these tumor immunogens imparted protection against parental tumor growth through activation of a specific immune response.