A new type of O-linked oligosaccharide has been discovered on several proteins secreted by the Gram-negative bacterium Flavobacterium meningosepticum, including Endo F2 (three sites), Endo F3 (one site), and a P40 protease (one site). The oligosaccharide moiety is covalently attached via a mannose residue to a serine or threonine at consensus sites corresponding to Asp-Ser* or Asp-Thr*-Thr. Preliminary characterization by mass spectroscopy revealed an oligosaccharide of 1244 Da at each of the proposed glycosylation sites. Collision-associated dissociation analysis showed a characteristic daughter ion series of m/z 218, 394, and 556, indicative of a common Flavobacterium oligosaccharide. Compositional analysis demonstrated an unusual profile of monosaccharides, including hexoses, methylated hexoses, and uronic acid derivatives.