Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism. We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E. coli Gcl and isolation of mutants lacking the enzyme. Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway. Significant sequence identity (26%) was also observed with E. coli pyruvate oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J. E., Jr. (1988) J. Bacteriol. 170, 3937-3945). Consistent with a grouping of Gcl with the AHAS and pyruvate oxidase enzymes. Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD. We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues. Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E. coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit. Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate.