Previous structural analysis of small oligosaccharide fragments had allowed the recognition of several small structural motifs within arabinogalactan, the dominant cell was structural polysaccharide of Mycobacterium tuberculosis. To determine how these motifs are connected to one another to form the complete polymer, oligosaccharide fragments containing up to 26 glycosyl residues were released by gentle acid hydrolysis of the per-O-methylated arabinogalactan, converted to fully per-O-alkylated oligoglycosylalditols, and purified by high-performance liquid chromatography, and the molecular weights and alkylation patterns of the resultant oligoglycosyl fragments were determined by fast atom bombardment mass spectrometry. The results, combined with previous studies, allowed further understanding of the intricate structural features of the nonreducing ends of arabinogalactan. Thus, the extended nonreducing ends of the arabinan were shown to consist of a tricosaarabinoside (23-mer). We reason that three such arabinan motifs are attached to the homogalactan component or arabinogalactan, which was previously shown to consist of alternating 5- and 6-linked beta-D-galactofuranosyl residues. Using the same approach as applied to the arabinan branches, an extended stretch of the galactan was isolated that consisted of at least 23 alternating beta-1,6 and beta-1,5 D-Galf residues, devoid of any branching, demonstrating that the points of attachment of the arabinan chains to galactan are close to the reducing end of galactan, which itself is linked to peptidoglycan via the linker disaccharide phosphate L-Rhap-(1-->3)-alpha-D-GlcNAc-P. By nuclear magnetic resonance analysis, the L-Rhap was shown to be in the alpha configuration. The long-chain alpha-alkyl-beta-hydroxy mycolic acids, known to occupy the 5-positions of both the terminal beta-D-Araf and internal 2-alpha-D-Araf residues of the terminal branched pentaarabinosyl motif, are now shown to be nonacylated at the beta-hydroxy function. Lack of acylation points to intramolecular hydrogen bonding between the beta-hydroxyl and carbonyl functions of the mycolic acid, providing a highly ordered arrangement of mycolic acids in accord with evolving models of the orientation of the cell wall polymers in mycobacterial cell walls. A revised model is proposed for the composition and orientation of the mycolyl-arabinogalactan in the cell walls of M. tuberculosis, which should increase our understanding of cell wall hydrophobicity, impermeability, and role in disease pathogenesis.