The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn5271 was determined. The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III. Expression studies were carried out with Alcaligenes sp. strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60. BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite. These mutants failed to grow on 3-Cba; however, they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently. These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite. Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate metaring fission pathway in Alcaligenes sp. BR60. The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x11)KHVLxEKPxA consensus flanked by alpha-helical domains. o-Phthalate cis-diol dehydrogenase (Pseudomonas putida), glucose-fructose oxidoreductase (Zymomonas mobilis), myo-inositol-2-dehydrogenase (Bacillus subtilis) and D-galactose-1-dehydrogenase (Pseudomonas fluorescens) are related proteins. These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III).