The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast. The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots. The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide. The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase. The substrate specificity of the recombinant enzymes expressed in S. cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.