Abstract: Recent evidence suggests that Paracoccidioides species have the potential to undergo sexual reproduction, although no sexual cycle has been identified either in nature or under laboratory conditions. In the present work we detected low expression levels of the heterothallic MAT loci genes MAT1-1 and MAT1-2, the alpha-pheromone (PBalpha) gene, and the alpha- and a-pheromone receptor (PREB and PREA) genes in yeast and mycelia forms of several Paracoccidioides isolates. None of the genes were expressed in a mating type dependent manner. Stimulation of P. brasiliensis MAT1-2 strains with the synthetic alpha-pheromone peptide failed to elicit transcriptional activation of MAT1-2, PREB or STE12, suggesting that the strains tested are insensitive to alpha-pheromone. In order to further evaluate the biological functionality of the pair alpha-pheromone and its receptor, we took advantage of the heterologous expression of these Paracoccidioides genes in the corresponding S. cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pbalpha pheromone either isolated from Paracoccidioides culture supernatants or in its synthetic form, both by shmoo formation and by growth and cell cycle arrests. This allowed us to conclude that Paracoccidioides species secrete an active alpha-pheromone into the culture medium that is able to activate its cognate receptor. Moreover, expression of PREB or PBalpha in the corresponding null mutants of S. cerevisiae restored mating in these non-fertile strains. Taken together, our data demonstrate pheromone signaling activation by the Paracoccidioides alpha-pheromone through its receptor in this yeast model, which provides novel evidence for the existence of a functional mating signaling system in Paracoccidioides.