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Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to biocontrol processes. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these proteases for industrial application. We report here a successful expression of the serine protease SL41 from a biocontrol fungus Trichoderma harzianum in Saccharomyces cerevisiae. A new serine proteases gene SL41 has been cloned from T. harzianum. The full-length cDNA was isolated by 5′ and 3′ rapid amplification of cDNA ends. The isolated cDNA of SL41 was then sequenced. The results showed that the open reading frame of SL41 was 1.617 bp long, encoding 538 amino acids. The cloning vector pMD18-T and an E. coli DH5α host were used to yield clones as E. coli DH5α/SL41. The SL41 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/SL41. Serine protease expressed by pYES2/SL41 was induced by galactose (maximal activity 16.5 units ml−1 at 40°C, pH 8.0) and was produced in fermentation liquid cultured for 60 h. Northern blot analysis indicated that SL41 transcripts are differentially accumulated at different culture time.