The dcuD gene (formerly yhcL) of Escherichia coli shows significant sequence similarity only to the dcuC gene of E. coli, which encodes a C4-dicarboxylate carrier (DcuC) that functions during anaerobic growth. Inactivation of dcuD had no effect on the growth of E. coli under a large number of conditions and led to no detectable changes in phenotype. Translational dcuD'-'lacZ gene fusions were not significantly expressed in the presence of dicarboxylates or monocarboxylates under oxic or anoxic conditions. Other potential substrates such as amino sugar derivatives, amino acids, and alpha-aspartyl dipeptides also did not lead to expression of dcuD. Changes in medium composition, pH, ionic strength, and temperature had no significant effects on dcuD expression. A dcuD gene amplified from a natural isolate of E. coli was not expressed in wild-type and E. coli K-12 backgrounds. Cloning of dcuD behind an inducible promoter resulted in the synthesis of a protein of the expected size (49 kDa), which, however, did not complement for the loss of DcuC or other C4-dicarboxylate carriers. It is suggested that dcuD encodes a protein of the DcuC family of anaerobic C4-dicarboxylate carriers and that dcuD is not significantly expressed or is expressed only under conditions not related to carboxylate metabolism. When two adjacent open reading frames (y0585 and y0586) from Haemophilus influenzae are fused, the resulting hypothetical protein has sequence similarity to DcuC and DcuD.