The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and 1 H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the α-pyranose-ring form of 7-desoxy-7-amino-d-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-α-d-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-d-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexadecasphingenin e. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.