The 14 nuo genes encode the subunits of the type I (energy-conserving) NADH dehydrogenase, a key component of the respiratory chain. Salmonella typhimurium, like Escherichia coli, has two enzymes that can oxidize NADH and transfer electrons to ubiquinone, but only the type I enzyme translocates protons across the membrane to generate a proton motive force. Cells with the type I enzyme are energetically more efficient; the role of the type II enzyme (encoded by ndh) is not established, but it may function like a relief valve to allow more rapid NADH recycling. Here, we have investigated transcription of the nuo gene cluster, primarily in S. typhimurium. Studies with polar insertion mutants demonstrate that these genes are arranged as a single, large operon that is expressed from a complex promoter region upstream of nuoA. The DNA sequence of the promoter region was determined, and primer extension analysis of nuo transcripts was used to map four major RNA 5' ends to this region. A set of lac operon fusions to various DNA segments from the nuo promoter region was also constructed. Analysis of these fusions confirmed the presence of at least two nuo promoters. Mutations in the global regulatory genes arcA, oxrA (fnr), crp, cya, and katF were tested for effects on expression of the nuo operon. However, none of the mutations tested had a large effect on expression of type I NADH dehydrogenase.