Abstract Endo-α- N-acetylgalactosaminidase from Alcaligenes sp. released the disaccharide, Galβ1→3GalNAc, from both dansylated serine-GalNAc-Gal and threonine-GalNAc-Gal, and showed higher activity on the former than the latter. The Km values were 0.17 mM and 1.43 mM with DNS-Ser-GalNAc-Gal and DNS-Thr-GalNAc-Gal, respectively. The optimum pHs were found to be 4.5–7.5 and 4.5–6.0 on DNS-Ser-GalNAc-Gal and DNS-Thr-GalNAc-Gal, respectively. On the contrary, the enzyme from Diplococcus pneumoniae had low activity to release the disaccharide from the amino acid- O-glycans. The possibility that the same O-glycoside but linked to different aglycon amino acids may play a different biological role in glycoproteins is discussed.