A neutral and an acidic polysaccharide with molecular masses of about 22 kDa and 45 kDa, respectively, were isolated from the N-acetylated cell walls of Bacillus subtilis AHU 1219 by heating at pH 2.5, followed by separation of the water-soluble product by ion-exchange chromatography and gel chromatography. The neutral polysaccharide, accounting for 40% of the mass of the cell walls, contained glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine in a molar ratio of 1:2:1:1. The minor, acidic polysaccharide contained glucuronic acid, glucose, galactose, L-serine and L-threonine in an approximate molar ratio of 1:1:1:0.5:0.5. Lysozyme digestion of the N-acetylated cell walls gave a polymer containing the neutral polysaccharide and glycopeptide components and another polymer which contained the acidic polysaccharide components together with small proportions of the neutral polysaccharide and glycopeptide components. Thus, the neutral and acidic polysaccharide chains seem to be attached to peptidoglycan through acid-labile linkages in the cell walls of this strain. Structural analysis of the neutral-polysaccharide preparation, involving 1H-NMR and 13C-NMR measurement, methylation and Smith degradation, led to the most likely structure, ----6)[Glc(beta 1----3)]GalNAc(alpha 1----4)-[GlcNAc(beta 1----3)]ManNAc(beta 1----4)GlcNAc(beta 1----, for the repeating units of this polysaccharide.