A serine protease produced by Thermomonospora fusca YX ( TfpA) is heat-stable ( up to 85 degrees C) and has a broad pH activity range and strong resistance to detergents. The objective of this study was to determine if the methylotropic yeast Pichia pastoris could express TfpA extracellularly. A 1.0- kb DNA fragment ( tfpA) encoding the pro-peptide and mature protein of TfpA was cloned into expression vectors pPICZ alpha A ( inducible) and pGAPZ alpha A ( constitutive) and introduced into P. pastoris by electroporation. Expression of r- TfpA was greater in the inducible system than in the constitutive one, producing 135 U ml(-1) medium supernatant 6 days after methanol induction. The r- TfpA was not glycosylated (21.7 kDa), and had pH and temperature optima of 8.5 and 80 degrees C, respectively, using azocasein as a substrate. In conclusion, P. pastoris can be used as a host to produce extracellular r- TfpA, and expression efficiency may be improved by optimizing fermentation conditions and modifying factors related to protein expression and stability.