The chlA encodes functions required for the biosynthesis of the molybdopterin part of the molybdenum cofactor. Mutants, carrying gene fusions at the chlA locus, which place β-galactosidase expression under the control of the chlA promoter, have been isolated employing λ plac Mui as the mutagen. The mutants exhibited β-galactosidase expression which was greatly enhanced when grown anaerobically. Secondary mutations at the chlB, D, E or G loci did not affect the high level of expression. The fnr gene product was not required for the anaerobic expression. Bacteriophage λ transducing phages were isolated which carried the φ (chlA-lac) mutations and were used to construct chlA/φ (chlA-lac) merodiploids. The merodiploids exhibited a much lower level of expression but showed the same characteristics as strains carrying lac fusions to the single chromosomal chlA locus. Genetic evidence is presented which strongly suggests that the molybdenum cofactor is a repressor of chlA expression. The anerobic enhancement of chlA expression is mediated via a mechanism that is distinct from the molybdenum cofactor effect.