The 3-O-methyl-D-mannose-containing polysaccharide (MMP) from Mycobacterium smegmatis, first described by Gray and Ballou (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) is now shown to be a mixture of at least four isomers separable by gel filtration owing to differences in size and degree of methylation. The major component is 3-O-methylmannose but all contain small amounts of mannose. The molecular weights range from 2040 to 2490 and all are nonreducing. After Smith degradation, all yield a single large and one or more small fragments that give 3-O-methylmannose as the sole product of complete acid hydrolysis. The large Smith-degraded MMP components (SD-MMP) are similar to intact MMP and vary from 1830 to 2130 daltons, consistent with the loss of a single mannose; whereas the smaller fragments are the size of tri- to hexasaccharides and result from fragmentation of incompletely methylated chains. Controlled acid hydrolysis of [methyl-3H]MMP releases 6% of the methyl groups as [3H]methanol at a rate characteristic for the hydrolysis of methyl alpha-D-mannopyranoside. Proton magnetic resonance spectra of MMP and SD-MMP show a major methyl ether proton peak and a second small peak at higher field equivalent to about one methyl group per molecule. The results are consistent with the presence of an alpha-methyl aglycon at the reducing end of the chains. Methylation analysis of MMP isomers purified by high pressure liquid chromatography confirms that they are linear and unbranched. Methylation of [methyl-3H]MMP yields unlabeled tetra-O-methylmannose, showing that the chains are terminated by mannose. However, digestion of [methyl-3H]MMP with alpha-mannosidase releases mannose and exposes [methyl-3H]3-O-methylmannose. Smith degradation of [methyl-3H]MMP III yields a penta-to hexasaccharide product that can be resolved by high pressure liquid chromatography into two components. The distribution of radioactivity between these two fragments suggests that the chain was cleaved near the middle and that there must be an unmethylated mannose at that position. We conclude that the 3-O-methylmannose polysaccharides are linear unbranched chains of 11 to 14 sugar units, each terminated by a single mannose at the nonreducing end and by a methyl aglycon at the reducing end. Each isomer shows microheterogeneity, with 1 or 2 unmethylated mannose units near the middle of some but not all of the chains.