The biosynthesis of alpha-N-acetylglucosaminidase in normal and Sanfilippo B fibroblasts was studied by labeling cells with [35S]methionine and isolation of the enzyme by immunoprecipitation. The immunoprecipitated polypeptides were separated by polyacrylamide gel electrophoresis and visualized by fluorography. alpha-N-acetylglucosaminidase is synthesized as a precursor of an apparent mol. wt. of 87,000. Intracellular processing of the precursor yields two polypeptides of apparent mol. wts. of 73,000 and 76,000 via several intermediates. It is accomplished within 3 days after synthesis. Less than 30% of the newly synthesized precursor is secreted. In the presence of 10 mM NH4Cl, secretion is enhanced to more than 80%. In our study, no alpha-N-acetylglucosaminidase polypeptides could be detected in fibroblasts from patients affected with either the severe or mild form of Sanfilippo disease, type B.