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Abstract: In this study, the isolation and characterization of Trichoderma virens glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) and its promoter is described. A cDNA clone of a partial GPD1 had been identified from an ongoing work on T. virens Expressed Sequence Tag (EST) analysis. This led to the isolation of a 2.9 kb T. virens GPD1 that encompasses the 5-regulatory flanking region (1,364 bp), open reading frame (1,448 bp) and 3-regulatory flanking region (31 bp) by DNA walking. Based on this sequence, a 1.017 kb cDNA fragment encompassing the Open Reading Frame (ORF) that encodes for GPD1 was subsequently isolated by reverse transcription-polymerase chain reaction. Comparison of the GPD1 and its cDNA sequences demonstrated that the complete gene sequence encodes a polypeptide chain of 338 amino acids interrupted by 2 introns. Sequence comparison analysis of the 5 non-coding region with the 5 flanking sequences of other fungal GPD genes show several regions of similar sequence. The segments from positions -68 bp relative to the start codon is potentially a transcription start site and is mapped within the pyrimidine rich region. The presumptive TATA and CAAT boxes are mapped at -363 to -358 and -109 to -105 from the initiation of translation sites, respectively. The deduced protein product is 71 to 96% identical to glyceraldehyde-3-phosphate dehydrogenases of other filamentous fungi. Phylogenetic analysis based on deduced amino acid sequences shows that GPD1 of T. virens forms a cluster with filamentous ascomycetes. The sequence of this gene and its promoter can be used for the development of genetic tools in molecular studies of T. virens and in the expression of heterologous genes.