A cDNA encoding a chaperone ClpA homologue of Paracoccidioides brasiliensis was isolated and characterized. The ClpA belongs to a group of ClpATPAses proteins, which are highly conserved, and include several heat inducible molecular chaperones. In this study, a 2879 bp cDNA designated as Pbclpa was obtained which encodes a predicted protein of 927 amino acids. Characteristic consensus motifs of the ClpATPases family are present. The PbClpA middle region was compared to other related ClpA and ClpB proteins from fungi and bacteria. Comparative analysis demonstrated in the middle region the presence of a heptad repeat sequence, characteristic of ClpBs from prokaryotes and fungi, which are absent in ClpAs from prokaryotes but were present in all described fungal ClpAs. Our comparative analysis reveals that one of the criteria typically used to distinguish the prokaryotic subfamilies ClpA and ClpB, the size of the middle sequence, may not be useful in fungi. Phylogenetic analyses were performed with the complete sequences of ClpAs from fungi and bacteria and with the middle regions of those ClpAs present at NCBI and Pfam databases. Our results indicated that both types of analysis can be useful as a tool in the determination of phylogenetic relationships.