During attempts to clone the gene coding for the respiratory NADH dehydrogenase of Escherichia coli, two hybrid plasmids were constructed from E. coli chromosomal DNA [Young, I. G., Jaworowski, A., & Poulis, M. I. (1978) Gene 4, 25-36]. One of these plasmids, pIY1, derived from EcoRI-digested chromosomal DNA, was studied in detail and shown to possess the gene coding for the NADH dehydrogenase of the aerobic respiratory chain of E. coli. We now report the characterization of the other hybrid plasmid, pIY2, derived from HindIII-digested chromosomal DNA, and shown that it complements ndh mutants not by virtue of carrying the ndh gene but because it carries the gene coding for the respiratory D-lactate dehydrogenase. Cells carrying this hybrid plasmid overproduce the respiratory D-lactate dehydrogenase in their cell membranes by 15-20-fold with negligible activity appearing in the cytoplasm. This results in an amplification of the levels of the D-lactate oxidase. The amplified D-lactate oxidase activity, coupled with the pyridine nucleotide linked D-lactate dehydrogenase, apparently provides a new pathway for the oxidation of reduced nicotinamide adenine dinucleotide (NADH) in the cell, independent of the respiratory NADH oxidase.