We have previously reported on purification and characterization of an exo-beta-D-glucosaminidase (Gls93) from culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetyl-D-glucosamine (GlcNAc). The corresponding gene of Gls93 was cloned and characterized in this work. To our knowledge, this is the first report on cloning of the gene encoding fungal exo-beta-D-glucosaminidase. This gene has no introns and encodes a polypeptide of 892 amino acids (aa) containing a secretion signal of 28 amino acids. Comparison of the amino acid sequence to known proteins and phylogenetic analysis indicated that gls93 belongs to the glycoside hydrolase family (GHF) 2 and should be further classified into a new subgroup, exo-beta-D-glucosaminidase subgroup. The gls93 transcription was biphasic when T. reesei was grown on GlcNAc, suggesting that the expression of this gene may be regulated by a complex mechanism, in which multiple regulatory proteins are involved. Furthermore, gls93 could be expressed in Pichia pastoris (ca. 0.49-mg/ml culture). The recombinant Gls93 had the two molecular forms, ca. 105 and 100 kDa, whose difference is caused by N-glycosylation. Both of them had the same properties such as specific activity and substrate specificity and showed only the activity of exo-beta-D-glucosaminidase but not those of beta-galactosidase, beta-glucuronidase, and beta-mannosidase belonging to GHF2.