Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in Latin America. The successful resolution of infection with this fungus is dependent on the activation of cellular immunity. We previously identified heat shock protein 60 (HSP60) as a target of the humoral response in paracoccidioidomycosis. Herein we expressed the gene encoding HSP60 in Escherichia coli and analyzed the immunological activity of this recombinant antigen. The immunization of BALB/c mice with recombinant protein emulsified in adjuvant stimulated a cellular immune response. Splenocytes from immunized mice proliferated in response to antigen and released interleukin-12 and gamma interferon (IFN-gamma). Vaccination with HSP60 reduced the fungal burden in mice given 10(6) or 10(7) yeasts and protected mice from a lethal challenge. The efficacy of the vaccination was blunted by the neutralization of IFN-gamma. CD4(+) cells were necessary for the efficacy of the vaccination in both the afferent and efferent phases. Thus, we have demonstrated that this immunodominant antigen is a candidate for the development of a vaccine against this fungus.