We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host-range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon omega failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK-driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were FixRed, but on Vigna unguiculata were Fix-. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose.