Structural studies were carried out on the linkage unit which joins ribitol teichoic acid to peptidoglycan in the cell walls of Lactobacillus plantarum AHU 1413. The heating of the cell walls at pH 2.5 led to release of only 5% of ribitol teichoic acid components as water-soluble material. In contrast, the same treatment of the cell walls after N-acetylation led to release of about 80% of the teichoic acid moiety, giving a teichoic-acid-linked sugar preparation which contained about equimolar amounts of mannosamine, glucosamine and glycerol as minor components. The teichoic-acid-linked sugar was hydrolyzed by mild alkaline treatment into a disaccharide, N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine and ribitol teichoic acid linked to glycerol. The Smith degradation of the N-acetylated cell walls gave a characteristic fragment, 1,2-ethylenediol-phospho-glycerol-phospho-N-acetylmannosaminyl(beta 1----4) N-acetylglucosamine. Furthermore, when the intact cell walls were subjected to the NaNO2 treatment followed by NaBH4 reduction, the ribitol teichoic acid moiety was recovered for the most part in the water-soluble polymer fraction, from which a sugar, N-acetylmannosaminyl-2,5-anhydromannitol, was released by mild alkaline treatment. These results lead to the conclusion that the ribitol teichoic acid chain in the intact cell walls of this organism is linked to peptidoglycan through a unique linkage unit, glycerol-phospho-N-acetylmannosaminyl(beta 1----4)-glucosamine. The anomalous stability of the linkage between the teichoic acid moiety and peptidoglycan against acid hydrolysis seems to be accounted for by the involvement of the N-substituted glucosamine residue in the phosphodiester bridge that joins the two polymers.