Background and aims : The acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum bv. trifolii is required for the establishment of effective symbiosis with compatible host plants (Trifolium spp.). In the rhizobium-legume interaction, early stages of root infection and nodule development have been well studied from a genetic standpoint. However, factors important for colonization of several surfaces by rhizobia, including soil particles and roots, have not yet been thoroughly investigated. The aim of this study was establishing of environmental factors affecting production of EPS by R. leguminosarum bv. trifolii strain 24.2 and the role of this polysaccharide in bacterial surface properties and attachment ability. ; Methods : Besides the wild-type strain, its derivatives differing in the level of EPS produced were used to these analyses. The ability of attachment to abiotic and biotic surfaces of these strains were established using CFU counting experiments. Three-dimensional structure and other parameters of biofilms formed were characterized in confocal laser scanning microscopy. Electrokinetic (zeta) potential of rhizobial cells were determined using Laser Doppler Velocimetry. ; Results : It was evidenced that the ability of R. leguminosarum bv. trifolii to produce EPS significantly affected bacterial attachment and biofilm formation on both abiotic and biotic surfaces. In addition, the presence of this polysaccharide influenced the zeta potential of rhizobial cells. Mutant strains having a mutation in genes involved in EPS synthesis were significantly impaired in attachment, whereas strains overproducing this polysaccharide showed higher adhesion efficiency to all of the tested materials. EPS facilitated attachment of bacterial cells to the tested surfaces most probably due to hydrophobic interactions and heterogeneity of the envelope surface. ; Conclusions : EPS produced by R. leguminosarum bv. trifolii plays a significant role in attachment and biofilm formation to both abiotic and biotic surfaces as well as bacterial surface properties.