Rhizobium leguminosarum bv. trifolii pssA encodes a glucosyl-isoprenylphosphate (IP)-transferase involved in the first step of exopolysaccharide (EPS) synthesis. It was found that the pssA gene is an important target for regulation of this biosynthetic pathway. The data of this study indicate that pssA transcription is a very complex and mainly positively regulated process. A detailed analysis of a 767-bp-long pssA upstream region revealed the presence of several sequence motifs recognized by regulatory proteins that are associated with phosphate-, carbon-, and iron-dependent regulation. In addition, numerous inverted repeats of different lengths have been identified in this region. pssA transcription is directed from two distal P1 and proximal P3 promoters whose sequences demonstrate a significant identity to promoters recognized by RNA polymerase sigma factor σ(70). Among rhizobial proteins, RosR seems to be a primary regulator that positively affects pssA expression. This protein binds to RosR box 1 located downstream of the P1 promoter. In addition, phosphate and the carbon source strongly affect pssA transcription. A significantly lower level of pssA expression was observed in both the wild-type strain growing under phosphate-rich conditions and the phoB mutant. In this regulation, the PhoB protein and Pho box 2 located upstream of the P3 promoter were engaged. pssA transcription is also significantly affected by glucose. Transcriptional analysis of a set of pssA-lacZ fusions expressed in Escherichia coli wild-type and cyaA and crp mutants confirmed that cyclic AMP (cAMP) receptor protein (CRP) and two cAMP-CRP boxes located upstream of the P1 are required for this upregulation. Moreover, the production of EPS was totally abolished in R. leguminosarum bv. trifolii mutant strains 4440 and 1012 containing a Tn5 insertion downstream of the P3 promoter and downstream of the P3 -35 hexamer, respectively.