The luminal surface of ileal enterocytes in fetal and neonatal mammals is covered by beta-hexosaminidase, which is attached to a fibrillar array. In this study, we have isolated this enzyme and subjected it to kinetic and structural analyses. The enzyme is identified as N-acetyl-beta-glucosaminidase (NA beta G) on the basis of substrate specificity and susceptibility to inhibition by N-acetylgalactosamine. Its catalytic properties and thermal stability are characteristics of the acidic, thermally labile human isoenzyme, but it differs from the human glycosidase in size. The neonatal NA beta G is a species of 225,000 relative mass (Mr), composed of two subunits of 125,000 and 115,000 Mr. Both its cellular location and differences in biophysical properties from the adult rat lysosomal forms and human glycosidases suggest that the neonatal rat NA beta G is a novel isoenzyme.