alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B, To study the human enzyme, we expressed its cDNA in Led mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300, It had an apparent K-m of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K-i = 0.45 mu M) and 6-acetamido-6-deoxycastanospermine (K-i = 0.087 mu M). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried, The purified enzyme preparation corrected the abnormal [S-35]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose B-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-N-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.