The cell wall arabinogalactans of strains of Mycobacterium, Rhodococcus, and Nocardia were per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides were reduced and per-O-ethylated to yield per-O-alkylated oligoglycosyl alditol fragments. Analyses of these fragments by gas chromatography-mass spectrometry and of the intact solubilized polysaccharides by H-1 and C-13 NMR revealed the major structural features of the different arabinogalactans from representatives of the different genera. All of the mycobacterial products contained a homogalactan segment of alternating 5-linked alpha-galactofuranosyl (Galf) and 6-linked beta-Galf residues. The arabinan segment consisted of three major domains, linear 5-linked alpha-arabinofuranosyl (Araf) residues and branched (3 --> 5)-linked Araf units substituted with either 5-linked Araf or the disaccharide beta-Araf-(1 --> 2)-alpha-Araf at both branched positions. The recognition of these features in in vivo grown Mycobacterium leprae is an important development. The arabinan from strains of Nocardia contains a nonreducing-end motif composed of the linear trisaccharide, beta-Araf-(1 --> 2)-alpha-Araf-(1 --> 5)-Araf, attached to linear 5-linked alpha-Araf units. The galactan segment of the arabinogalactan of Nocardia sp. is composed of linear 5-linked beta-Galf units substituted in part at O-6 with terminal beta-glucosyl units. The two representative strains of Rhodococcus also differed in the composition of the galactan moiety; in addition to the 5-linked Galf, 2- and 3-linked beta-Galf units are present. The reducing end of the galactans, and therefore, apparently, of the entire arabinogalactans from all species from all genera, are apparently composed of the unit, rhamnosyl-(1 --> 3)-N-acetyl-glucosamine, which, in turn, is apparently attached to peptidoglycan via phosphodiester linkage.