Abstract The polysaccharide containing 6-O-methylglucose, from Mycobacterium phlei, is an acidic molecule with at least seven 6-O-methyl-d-glucopyranosyl residues linked α-1,4', and with a side chain of at least three α-1,4'-d-glucopyranosyl units. A simplified procedure is described for isolating the polysaccharide (MGP) from acetone-dried M. phlei cells with improved yield. Exhaustive α-amylase digestion of the polysaccharide released glucose, maltose, and a disaccharide. The last yielded d-glucose and 3-O-methyl-d-glucose after acid hydrolysis; while hydrolysis, after reduction with sodium borohydride, gave glucitol and 3-O-methylglucose. Methylation analysis established that the disaccharide was 3-O-methyl-α-d-glucopyranosyl-(1 → 4)-d-glucose. Limited α-amylolysis of MGP yielded a trisaccharide and a tetrasaccharide, each containing 1 mole of 3-O-methylglucose at the nonreducing end. The trisaccharide was digested by α-amylase to the disaccharide 3-O-methylglucosyl-(1 → 4)-glucose and glucose, while α-amylolysis of the tetrasaccharide gave these products plus maltose. A glucoamylase preparation converted the tetrasaccharide to the trisaccharide and glucose. That 3-O-methylglucose was at the nonreducing terminus of each saccharide was shown by partial acid hydrolysis studies and by the products of exhaustive propylation, which yielded 3-O-methyl-2,4,6-tri-O-propylglucose. Propylation of the intact polysaccharide and analysis of the fully alkylated methylglucosides by gas-liquid chromatography established that 3-O-methylglucose and glucose were in the nonreducing terminal positions. After α-amylase digestion, glucose was the only terminal residue. Glucoamylase removed an additional glucosyl residue, which had been linked α-1,4' to a 6-O-methylglucosyl residue. An organic acid having the chromatographic and electrophoretic properties of glyceric acid was isolated from acid hydrolysates of MGP. The glyceric acid was oxidized by 1 mole of sodium periodate, with the release of 1 mole of formaldehyde and 1 of glyoxalate. It was shown to have the d configuration. 3-O-Methylglyceric acid was obtained by hydrolysis of methylated MGP, showing that the linkage to the polysaccharide involved the 2-hydroxyl group. MGP was converted to the hydroxamate, and a Lossen rearrangement was performed in water at neutral pH. The reducing sugar thus formed was reduced with sodium borotritide, and the radioactive polyol obtained by acid hydrolysis was identified as glucitol. Thus, the glyceric acid must be attached glycosidically to a glucose residue. The intact lipopolysaccharide, MGLP, was not associated with purified M. phlei cell wall or particulate preparations. The material was recovered quantitatively from the high speed supernatant, apparently in free form. An attempt to degrade the main chain enzymatically with extracts from M. phlei was unsuccessful, and a search both for low molecular weight oligosaccharides containing 6-O-methylglucose and for large polysaccharides into which MGLP might have been incorporated was unproductive. Thus, our investigations have failed to suggest a structural or metabolic function for the lipopolysaccharide. MGLP, isolated from the cultural filtrate of M. phlei cells in the stationary phase, was indistinguishable from the intracellular molecule. It was released into the medium during all stages of the growth cycle, the kinetics following those for the appearance of low molecular weight carbohydrates and of mannose- and arabinose-containing polysaccharides.