A cDNA coding for an antigenic protein (rPb27) from the pathogenic fungus Paracoccidioides brasiliensis was cloned and its protective activity was determined against paracoccidioidomycosis (PCM). The cDNA sequence contained an open reading frame (ORF) of 660 base pairs encoding a protein of 219 amino acids with a predicted molecular weight of 25kDa. The deduced amino acid sequence exhibited 100% identity to the 27kDa P. brasiliensis hypothetic protein (access number AA49615). The complete coding cDNA was cloned into a pGEX 4T-2 plasmid and expressed in Escherichia coli as a glutathione-S-transferase-tagged (GST) recombinant protein. Mice immunized with purified rPb27 were able to develop high levels of IgG2b, moderate levels of IgG1 and low levels of IgG2a. At the same time the levels of TGF-beta and IFN-gamma were high while a very low production of IL-10 was verified. Using confocal microscopy with anti-rPb27 mouse serum against P. brasiliensis yeast forms, surface and cytosolic staining pattern were observed. Moreover, immunization of mice with this antigen induced a significant degree of protection in the lungs (93%), liver (93%) and spleen (100%) at 60 days after challenge with infection. Thus, the granulomatous lesions revealed a greater degree of compaction and organization, with few lesions in the lungs and no dissemination of the fungus to other organs. These results showed that a recombinant protein of P. brasiliensis (rPb27) promoted acquired protection against infection with P. brasiliensis yeast forms, suggesting the use of this protein for future development as a prophylactic vaccine for PCM.