Three enzymes are specifically required for uptake and catabolism of citrate by Klebsiella pneumoniae under anaerobic conditions: a Na+-dependent citrate carrier (CitS), citrate lyase (CitDEF), and the Na+ pump oxalo-acetate decarboxylase (OadGAB). The corresponding genes are clustered on the chromosome, with the citCDEFG genes located upstream and divergent to the citS—oadGAB genes. We found that expression of citS from its native promoter in Escherichia coli requires the DNA region downstream of oadB. Nucleotide sequence analysis of this region revealed the presence of two adjacent genes, citA and citB, By sequence similarity, the predicted CitA and CitB proteins were identified as members of the two-component regulatory systems. The sensor kinase CitA contained, in the N-terminal half, two putative transmembrane helices which enclosed a presumably periplasmic domain of about 130 amino acids. The C-terminal half of the response regulator CitB harboured a helix-turn-helix motif typical of DNA-binding proteins. K. pneumoniaecitB null mutants were unable to grow anaerobically with citrate as the sole carbon and energy source (Cit− phenotype). When cultivated anaerobically with citrate plus glycerol, all of the citrate-specific fermentation enzymes were synthesized in the wild type, but not in the citB mutants. This showed that citS, oadGAB and citDEF required the CitB protein for expression and therefore are part of a regulon. In the wild type, synthesis of CitS, oxalo-acetate decarboxylase and citrate lyase was dependent on the presence of citrate, sodium ions and a low oxygen tension. In a citA null mutant which expressed citB constitutively at high levels, none of these signals was required for the formation of the citrate fermentation enzymes. This result suggested that citrate, Na+, and oxygen exerted their regulatory effects via the CitA/CitB system. In the presence of these signals, the citAB gene products induced their own synthesis. The positive autoregulation occurred via co-transcription of citAB with citS and oadGAB.