A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).