The dcuA and dcuB genes of Escherichia coli encode homologous proteins that appear to function as independent and mutually redundant C4-dicarboxylate transporters during anaerobiosis. The dcuA gene is 117 bp downstream of, and has the same polarity as, the aspartase gene (aspA), while dcuB is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene (fumB). To learn more about the respective roles of the dcu genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy dcuA- and dcuB-lacZ transcriptional fusions. The results show that dcuA is constitutively expressed whereas dcuB expression is highly regulated. The dcuB gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition, dcuB is strongly induced by C4-dicarboxylates, suggesting that dcuB is under the control of an uncharacterized C4-dicarboxylate-responsive gene regulator. Northern blotting confirmed that dcuA (and aspA) is expressed under both aerobic and anaerobic conditions and that dcuB (and fumB) is induced anaerobically. Major monocistronic transcripts were identified for aspA and dcuA, as well as a minor species possibly corresponding to an aspA-dcuA cotranscript. Five major transcripts were observed for dcuB and fumB: monocistronic transcripts for both fumB and dcuB; a dcuB-fumB cotranscript; and two transcripts, possibly corresponding to dcuB-fumB and fumB mRNA degradation products. Primer extension analysis revealed independent promoters for aspA, dcuA, and dcuB, but surprisingly no primer extension product could be detected for fumB. The expression of dcuB is entirely consistent with a primary role for DcuB in mediating C4-dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.