We have detected an endoglycosidase activity produced by Flavobacterium meningosepticum. This enzyme, named endo F, cleaves glycans of both the high-mannose and the complex type linked through asparagine to the protein backbone. The data indicate that cleavage occurs via hydrolysis of the gly-*cosidic bond ofthe N,N'-diacetylchitobiose core structure adjacent to asparagine, similar to that due to endo H and endo D. Extreme variability was noted in the availability of this cleavage site among N-linked glycoproteins. Glycoproteins of retrovirus, lymphocytic choriomeningitis virus, Pichinde virus, and.HLA-A and -B antigens were readily cleaved in the presence of nonionic detergent.Others, such as ovalbuminjfetuin, bromelain,.ovomucoid, al-acid glycoprotein, immunoglobulin G, and influenza virus hemagglutinin became susceptible only after reduction and alkylation or when cleavage was performed in the presence of 1% 2-mercaptoethanol. Endo F should prove useful in the study of glycans and protein backbones as' discrete entities. and for defining the nature of the glycan-protein interface.Specific glycosidases have proven useful in elucidation of the structures and biosynthetic pathways of biologically important glycoproteins (1-5). Two of the most useful are the endoglycosidases endo H (1) and endo D (2), which cleave high-mannose and complex glycans, respectively, from glycoproteins and glycopeptides. Cleavage by these enzymes occurs between the adjacent N-acetylglucosamine residues linking the glycan moiety to the asparagine of the protein backbone (1, 2). In this report, we describe an endoglycosidase that cleaves both comvplex and high-mannose glycans from asparagine-linked glycoproteins.The enzymatic activity. was first detected in a commercial preparation of carboxypeptidase A.* Examination of this reagent revealed considerable bacterial contamination and one of the bacterial species released endoglycosidase.activity into its culture fluid. The bacteria have been typed as Flavobacterium meningosepticumt (6). In keeping with previous endoglycosidase nomenclature (2) .The cells were removed from the culture medium by centrifuigation at 5,000 x g for 10 min. The culture supernatant was concentrated by addition of solid ammonium sulfate to 80% saturation,.followed by centrifugation after stirring for 1 hr at 4°C. The concentrated protein was suspended in 50% ammonium sulfate. The suspension was centrifuged, and the pellet was dissolved in 0.01 M sodium phosphate, pH 7.2/0.15 M NaCl (Pi/ NaCl)/50 mM EDTA. This material was chromatographed on a column (1.5 x 50 cm) of ACA54 (LKB, Uppsula, Sweden) at .40C in Pi/NaCV5 mM EDTA. The peak fractions were pooled -and 'concentrated to 1/10 vol in a dialysis bag against dry Sephadex G-100. The material was made 50% glycerol and stored at -20°C. Endo F remains active under these conditions for at .least.3 months.Several attempts by ion-exchange' chromatography failed to physically separate distinct glycosidase activities. Isoelectric focusing in agarose followed by el...