beta-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-beta(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a beta-hexosaminidase from Streptomyces plicatus to 2.2 A resolution (Protein Data Bank code ). beta-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogue N-acetylglucosamine-thiazoline and beta-hexosaminidase from S. plicatus to 2.1 A resolution (Protein Data Bank code ). Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a (4)C(1) chair. A tryptophan-lined hydrophobic pocket envelopes the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 beta-hexosaminidases.