Megaplasmid pSymB of the nitrogen-fixing symbiont Sinorhizobium meliloti, implicated in adaptation to hyperosmotic stress, contains 11 gene clusters that apparently encode surface polysaccharides. However, only 2 of these clusters, containing the exo and exp genes, have been associated with the synthesis of the acidic exopolysaccharides succinoglycan and galactoglucan, respectively. The functions of the other 9 clusters remain unsolved. The involvement of one of those regions, pSymB cluster 3, on surface polysaccharide synthesis and its possible implication in osmoadaptation were investigated. In silico analysis of cluster 3 showed that it putatively encodes for the synthesis and transport of a methylated surface polysaccharide. Mutants affected in this cluster were symbiotically effective but showed defects in growth under saline and nonsaline osmotic stress. The gene SMb21071, encoding a putative initiating glycosyltransferase, is transcriptionally induced under hyperosmotic conditions. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and silver staining showed that osmotic stresses changed the profiles of surface polysaccharides of wild-type and mutants strains in different ways. The overall results suggest that cluster 3 is important for growth under saline stress and essential for growth under nonsaline hyperosmotic stress, and it appears to be implicated in maintaining and (or) modifying surface polysaccharides in response to osmotic stress.