Aspergillus nidulans produces several proteases. The prtA gene encodes a major protease, and two approaches were explored to achieve the overproduction of this enzyme. Molecular cloning of the mature form of this enzyme in Pichia pastoris resulted in the production of an inactive form. In addition, the presence of this enzyme was toxic for the host and resulted in cell lysis. The modification of the culture medium constituents resulted in a 6.4-fold increase in enzyme production. The main effect was achieved through the use of organic nitrogen sources. Although it was previously shown that the PrtA protease shows promiscuous esterase activity, the production of this enzyme was not induced by lipidic sources.