Chemical modification in combination with site-directed mutagenesis was used to identify a tyrosine residue responsible for the increase in ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) nucleotidase activity after acetylation with a tyrosine-selective reagent, N-acetylimidazole. The NTPDase3 ATPase activity is increased more than the ADPase activity by this reagent. Several fairly well conserved tyrosine residues (252, 255, and 262) that are located in or very near apyrase conserved region 4a (ACR4a) were mutated. These mutants were all active, but mutation of tyrosine 252 to either alanine or phenylalanine eliminated the activity increase observed after N-acetylimidazole treatment of the wild-type enzyme. This suggests that the acetylation of tyrosine 252 is responsible for the increased activity. Stabilization of quaternary structure has resulted in increased enzyme activities for the NTPDases. However, mutation of these three tyrosine residues did not result in global changes of tertiary or quaternary structure, as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis. Nevertheless, disruption of the oligomeric structure with the detergent Triton X-100 abolished the increase in activity induced by this reagent. In addition, mutations that abolished the N-acetylimidazole effect also attenuated the increases of enzyme activity observed after lectin and chemical cross-linking treatments, which were previously attributed to stabilization of the quaternary structure. Thus, we speculate that the acetylation of tyrosine 252 might induce a subtle conformational change in NTPDase3, resulting in the observed increase in activity.