Four oligosaccharide chain-cleaving enzymes, including two new endoglycosidases distinct from endo-beta-acetylglucosaminidase (Endo) F1, have been identified and purified to homogeneity from cultural filtrates of Flavobacterium meningosepticum. FPLC-directed hydrophobic-interaction chromatography in conjunction with high-resolution ion-exchange chromatography provided a more simple, rapid method for the isolation of endoglycosidase F1, F2 and F3, and the amidase, peptide-N4-N-acetyl-beta-D-glucosaminyl)-asparagine amidase (PNGase F), in greater than 50% yield. The specificity of PNGase F and Endo F1 are well established. Endo F2 and Endo F3 represent new distinct endoglycosidases that prefer complex as compared to high-mannose asparagine-linked glycans. Endo F2 cleaved biantennary oligosaccharides, whereas Endo F3 cleaved both bi- and triantennary oligosaccharides.