Exopolysaccharide production by Rhizobium leguminosarum bv. trifolii is required for successful establishment of nitrogen-fixing symbiosis with clover (Trifolium pratense L.). Using plasmid-borne transcriptional fusions of promoters of pss genes with promoterless lacZ the effect of root exudate, phosphate, and ammonia on expression of pssT, pssN, pssO, and pssP genes in wild-type strain RtTA1 background was determined. A stimulating effect of these environmental factors on pssO and pssP gene expression was observed. The putative pssO gene promoter was determined to be a strong promoter within which the divergent nod-box element was identified. The pssO promoter was slightly inducible in a flavonoid-dependent manner in wild-type R. leguminosarum bv. trifolii strains RtTA1 and ANU843 and very weakly active in a mutant of strain ANU843 that lacks the regulatory nodD gene. The expression of pssO and pssP genes in planta was investigated using plasmid-borne pssO-gusA and pssP-gusA fusions under different phosphate availability to clover. The level of pssO-gusA fusion expression was shown to be dependent on phosphate concentration in the plant growth medium.