Abstract A polysaccharide composed of 3-O-methyl-d-mannose and d-mannose in the molar proportion of 10:2 has been isolated from Mycobacterium phlei. This methylmannose polysaccharide (MMP) did not contain acidic or basic groups, acyl esters, or a reducing end. MMP had a molecular weight of 2084 ± 35 as determined by sedimentation equilibrium and had a partial specific volume of 0.663 cc per g as found both by sedimentation equilibrium and summation of the molal volumes of the hexose residues. The structure of MMP was investigated by methylation analysis, Smith degradation, and immunochemistry. In order to distinguish between mannose and 3-O-methylmannose residues in the polysaccharide after exhaustive methylation, MMP was isolated from cells grown in the presence of l-[methyl-14C]methionine, under conditions which resulted in the incorporation of radioactivity exclusively into the methyl groups of the 3-O-methylmannose residues. Exhaustive methylation of [methyl-14C]MMP with nonradioactive methyl iodide and hydrolysis of the fully methylated polysaccharide yielded radioactive 2,3,6-tri-O-methylmannose as the major component. Radioactive 2,3-di-O-methylmannose, and nonradioactive 3,4,6-tri-O-methylmannose and 2,3,4,6-tetra-O-methylmannose were obtained as minor components. These findings showed that all of the 3-O-methylmannose residues were (1 → 4)-linked with 1 residue containing a substituent at position 6 as well. One of the two mannose residues was (1 → 2)-linked and the other occupied a terminal position in the polysaccharide. Smith degradation of MMP converted the 2 mannose residues to glycerol and ethylene glycol, yielding a polysaccharide containing only 3-O-methylmannose. Exhaustive methylation of Smith-degraded [methyl-14C]MMP and hydrolysis of the fully methylated polysaccharide yielded radioactive 2,3,6-tri-O-methylmannose as the major component, although a small quantity of radioactive 2,3-di-O-methylmannose and a trace of radioactive 2,3,4,6-tetra-O-methylmannose were still detected. With some slight discrepancies, these results indicate that the ten 3-O-methylmannose residues are (1 → 4)-linked and occur in a homologous chain which probably forms a macrocyclic ring. The 2 mannose residues are attached to each other in a (1 → 2)-linkage and occur as a nonreducing end of the polysaccharide, or as a side chain on the postulated macrocyclic ring. Further evidence in support of a (1 → 2)-linked mannobiose side chain was provided by testing MMP as an inhibitor of the precipitin reaction between Kloeckera brevis phosphomonoester mannan and antisera obtained against whole cells of the yeast. MMP and α-(1 → 2)-linked mannotriose were equally effective as inhibitors of this precipitin reaction, which is specific for α-(1 → 2)-linked mannose residues at a terminus. Smith-degraded MMP, which had the mannobiose side chain removed, failed to inhibit the precipitin reaction. These results confirm the presence of at least 2 α-(1 → 2)-linked mannose residues at a terminal position of MMP.