Exoglycosidases modify carbohydrate epitopes on glycoproteins and glycolipids. The N-acetyl-alpha-D-galactosaminidase from the domestic chicken, Gallus domesticus, is an important exoglycosidase which degrades the human blood group A epitope. This enzyme has never been demonstrably purified or thoroughly characterized. We have developed a technique to purify this enzyme to homogeneity. The isolated enzyme has a molecular weight of 49.1 kDa by SDS PAGE and 145.0 kDa by gel filtration. The enzyme is highly selective for PNP-N-acetyl-alpha-D-galactosaminide and is inactive against other low molecular weight substrates. The enzyme hydrolyzes the terminal N-acetyl-alpha-D-galactosaminide residues from blood group A2 erythrocytes. Protease activity is below detectable limits. The enzyme has a pH optima of 3.7, a pI of 8.15, is relatively unaffected by ionic strength, and is stable at 4 degrees C.