The activity of Taka-N-acetyl-β-D-glucosaminidase [EC 3.2.1.30] toward fifteen newly synthesized substrate analogs with different N-substituent was determined. The contribution of the substituent constants of the substrate N-acyl group (Vw, σ*, and π) to the relative rate (FR) of enzymatic hydrolysis was evaluated quantitatively by regression analysis to ascertain the role of the substrate 2-acetamide group in the N-acyl specificity of this enzyme. The following equation gave the best fit for each parameter. A mechanism for the enzymic hydrolysis was proposed based on this relationship.