Mutants of the nadI and pnuA genes were independently isolated on the basis of defects in repression of NAD biosynthetic genes and defects in transport nicotinamide mononucleotide (NMN). The mutations map at min 99 on the Salmonella chromosome, and the affected regions appear to be cotranscribed. Some pairs of nadI and pnuA mutations complement, suggesting the existence of independent functions. However, cis/trans tests with particular mutations provide evidence that both repressor and transport functions are actually performed by a single bifunctional protein. (This result confirms sequencing data of Foster and coworkers [J. W. Foster, Y. K. Park, T. Fenger, and M. P. Spector, J. Bacteriol. 172:4187-4196, 1990]). We have designated the gene for this bifunctional protein nadI and distinguish the regulatory and transport defects with phenotypic designations (R and T). When a nadI(R- T+) mutation (eliminating only repression function) is placed cis to a superrepressor mutation, nadI(R(s) T-), the superrepression phenotype is lost. In contrast, placement of R- and R(s) T- mutations in trans allows full superrepression. This result suggests that the transport function (eliminated by the R(s) T- mutation) and the repression function are provided by the same protein. Insertion mutations in the promote-proximal repressor region of the nadI gene eliminate transport function unless the inserted element can provide both for both transcription and translation start signals; this finding suggests that there is no transcriptional or translation start between the regions encoding repression and transport functions.