The 65-kDa heat-shock protein (HSP65) is thought to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. We investigated the biological function and expression mechanism of HSP65 in macrophages of mice infected with Trypanosoma cruzi. BALB/c mice, which are susceptible to T. cruzi, showed high levels of parasitemia, and 80% of these mice died within 42 days after the infection, whereas resistant C57BL/6 or DBA/2 mice showed low levels of transient parasitemia and all survived. HSP65 expression was correlated with resistance to T. cruzi infection; HSP65 was more strongly expressed in macrophages of resistant C57BL/6 and DBA/2 mice than in macrophages of susceptible BALB/c mice. Immunodeficient BALB/c-nu/nu (nude) and C.B-17 scid/scid (SCID) mice were shown to be highly susceptible to this infection, and they did not express detectable levels of HSP65, suggesting that T cells play essential roles in the expression of HSP65 as well as in protective immunity against the infection. CD4(+) T cells, but not CD8(+) T cells or gammadelta T cells, were the cell population responsible for the induction of HSP65 expression in macrophages. Furthermore, depletion of asialo GM-1(+) NK cells made resistant C57BL/6 mice more susceptible to the infection, and HSP65 expression in their macrophages was abolished. Semiquantitative reverse transcription PCR analyses showed that both interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) mRNA levels in CD4(+) T cells became low when resistant C57BL/6 mice were depleted of NK cells, suggesting that NK cells contribute to functional differentiation of CD4(+) T cells and thereby affect the induction of HSP65 expression. To determine the function of HSP65, macrophages were treated in vitro with antisense oligonucleotide for HSP65 prior to inducing HSP65 with IFN-gamma plus TNF-alpha or T. cruzi infection. This treatment did not affect the production of nitric oxide following activation, but the treated macrophages became susceptible to apoptosis. These results indicate that HSP65 plays a role in preventing the apoptosis of macrophages and thereby contributes to host resistance against T. cruzi infection.