Microbial proteases play an essential role in scientific research and commercial applications. This study is to clone, sequence, and express a thermostable protease gene from the thermophilic fungi Chaetomium thermophilum and to generate yeast strains expressing C. thermophilum protease suitable for industrial applications. Degenerate primers were designed based on the conserved domain of other identified serine proteases and cDNA fragment of C. thermophilum gene pro was obtained through reverse transcriptase-polymerase chain reaction (RT-PCR). The full-length cDNA of 2007 bp was generated using RACE amplification. The cDNA contains an open reading frame of 1596 bp encoding 532 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the subtilisin serine proteases. The C. thermophilum gene pro was expressed in Escherichia coli BL21 (DE3) and Pichia pastoris, respectively and soluble protein was obtained in P. pastoris. The expressed protease was secreted into the culture medium by the yeast in a functional active form and the purified recombinant protease exhibits optimum catalytic activity at pH 8.0 and 60 degrees C. The enzyme is stable at 60 degrees C. The integration of gene pro into P. pastoris genome is stable after 10 generations and the yeast transformants showed a consistent protease expression. Gene pro encoding a serine protease from C. thermophilum was cloned, sequenced, and overexpressed successfully in P. pastoris. The expressed protease was purified and the properties of the recombinant protease are characterized. Chaetomium thermophilum is a soil-borne thermophilic fungus and the protease cloned from it is stable in a high temperature and a wide rage of pH. The overexpression of the enzyme in a mesophilic micro-organism offers a potential value for scientific research and commercial applications.