A gene encoding the entire highly expressed protein previously identified in the proteome of Paracoccidioides brasiliensis yeast cells as PbY20 has been isolated. The pby20 sequence reveals an open reading frame of 1364 bp and a deduced amino acid sequence of 203 residues, which shows high identity to benzoquinone reductase from Phanerochaete chrysosporium (72.0%), Saccharomyces cerevisiae Ycp4 (65%), and Schizosaccharomyces pombe p25 (59%), and to allergens from Alternaria alternata Alt a7 (70%) and from Cladosporium herbarum, Cla h5 (68%). Low levels of the pby20 transcript in the mycelium and highly induced ones in infective yeast cells during the transition of this dimorphic fungus indicate transcriptional control of its expression. PbY20 was immunologically detected only in yeast cell extract, suggesting an important role in cell differentiation or even in the maintenance of the yeast form. Immunoelectron microscopy showed that PbY20 is found inside large granules and vacuoles, in the nucleus, and also in the cytoplasm. Through sequence comparisons analysis and fluorescence emission assay, PbY20 was recognized as a member of the flavin mononucleotide flavodoxin-like WrbA family, which are involved in heat shock and oxidative stress in biological systems. Assuming that PbY20 belongs to this family, a similar role could be attributed to this protein.