O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked beta-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63-342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K(m) value and catalytic efficiency (k(cat)/K(m)) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K(m) value of 0.26 mM and the highest catalytic efficiency of 3.51.10(3). These results reveal that the N-terminal region (a.a. 1-350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.