The nucleotide sequence of the structural gene coding for the respiratory NADH dehydrogenase of Escherichia coli has been determined by the chain-termination method. The reading frame for the protein starts with the unusual initiation codon UUG and predicts an amino acid sequence of 434 residues (Mr = 47 304). The reading frame was confirmed by protein chemical studies including determination of the N-terminal sequence of the protein. The product made in vivo was found to have threonine as its N-terminal residue, indicating that the initiating N-formylmethionine had been removed by post-translational processing.