D-Arabino-D-galactan, the main antigenic polysaccharide of Mycobacterium species, was isolated from the cell walls of M. tuberculosis strain H37R5 and Aoyama B, M. bovis BCG, M. phlei and M. smegmatis, and also atypical Mycobacterium P1. All arabinogalactan preparations had similar chemical properties ([α]D+23.5–28°; ratio of D-arabinose to D-galactose, 5:2), and were immunologically identical. They have ramified structures, with repeating units of 11–16 sugar residues, consisting of °-{1→5)- and (1→2)(minor)-D-arabinofuranosidic linkages, and β-(1→4)-D-galactopyrano-sidic (or (1→?5)-furanosidic) linkages; the side chains are terminated with arabino-furanose residues and are attached to the main chains at C-3 of arabinose and probably at C-6 of galactose residues, respectively. The action of bacterial M-2 enzyme on the arabinogalactan of M. phlei resulted in the release of a series of D-arabino-oligosaccharides, which included α-(1→5)-linked arabinobiose, -triose, and -tetraose, leaving a degraded polysaccharide (ratio of arabinose to galactose, 2 : 5.4) which may represent a back-bone chain. The inhibitory activities of these oligosaccharides in the precipitin reaction between the arabinogalactan and the anti-cell-wall sera indicate that the arabinofuranosyl side chains are responsible for the serological activity of the- cell-wall polysaccharides. The arabinogalactan preparations reacted not only with antisera against mycobac-terial cell walls but also with those against Corynebacterium diphtheriae and Nocardia asteroides, suggesting that the arabinogalactan is a common antigen of these bacterial groups.