<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.</p> <p>Results</p> <p>A cDNA (<it>Pb</it>sp) encoding a secreted serine protease (<it>Pb</it>SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant <it>Pb</it>SP was produced in <it>Escherichia coli</it>, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the <it>P. brasiliensis </it>proteome. <it>In vitro </it>deglycosylation assays with endoglycosidase H demonstrated that <it>Pb</it>SP is a <it>N</it>-glycosylated molecule. The <it>Pb</it>sp transcript and the protein were induced during nitrogen starvation. The <it>Pb</it>sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of <it>Pb</it>SP with <it>P. brasiliensis </it>proteins were evaluated by two-hybrid assay in the yeast <it>Saccharomyces cerevisiae</it>. <it>Pb</it>SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2.</p> <p>Conclusions</p> <p>A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. <it>Pb</it>SP interactions with other <it>P. brasiliensis </it>proteins were reported. Proteins interacting with <it>Pb</it>SP are related to folding process, protein trafficking and cytoskeleton reorganization.</p