The cellular levels of the three translational initiation factors, IF1, IF2, and IF3, increase as a function of growth rate in parallel with those of ribosomes. Therefore both ribosomal and initiation factor gene expression is under metabolic control. To address how expression of the Escherichia coli gene for IF1, infA, is regulated, a 3-kilobase region of the genome surrounding infA was sequenced. The 5' and 3' termini of in vivo infA transcripts were defined by S1 nuclease mapping, and mRNA size was measured by Northern blot hybridization. The infA gene is transcribed by two promoters, P1 and P2, which generate transcripts of 525 and 330 nucleotides, apparently ending at the same rho-independent terminator. Analyses of operon and protein fusions to lacZ demonstrate that neither infA transcription nor translation is affected by high cellular levels of IF1. However, P2, but not P1, increases in activity as a function of the growth rate of the cell and is the dominant promoter in rich medium. Therefore, metabolic control of infA expression occurs exclusively at the level of transcription by the P2 promoter.