The basic structures of the extracellular polysaccharides of Rhizobium leguminosarum and Rhizobium trifolii were found to be identical, but their acylation patterns differ. Liquid hydrogen fluoride at -40 degrees degrades the two polysaccharides to a series of oligosaccharides representing the repeating units of the polysaccharides and their higher homologs. At -23 degrees, it degrades the polymers to a mixture of oligosaccharides from which a tetrasaccharide constituting a unit of the backbone of the polysaccharide, and a trisaccharide representing all but the non-reducing terminus of the side chain, could be readily purified. The location and identity of the acyl substituents were determined by 1H-n.m.r. spectroscopy, methylation analysis, and f.a.b. mass spectrometry. The unusual substituent D-3-hydroxybutanoate was found esterified to O-3 of a terminal 4,6-O-pyruvic acetylated D-galactose in both strains of R. leguminosarum, and in one of the three strains of R. trifolii tested. All of the strains tested contained a 3-O-acetyl substituent on the (1----4)-beta-D-glucopyranosyl residues in the backbone of the polysaccharide. Only the R. leguminosarum polysaccharides contained a combination of 2- and 3-O-acetyl groups on the branching sugar of the backbone of the polymer.