A detailed restriction site map was determined for an 8.4-kilobase DNA fragment containing the 5' regulatory and promoter region of the nar operon of Escherichia coli. The 5' end of the nar operon was subcloned as a 2.5-kilobase fragment, and an intact nar operon was constructed from this subcloned fragment and an EcoRI fragment containing the remainder of the nar operon. A set of Bal 31 deletions extending into the 5' region of the intact operon was selected, mapped, and characterized. Based on the synthesis of the alpha and beta subunits of nitrate reductase in a nar::Tn5 mutant, three categories of deletions were found: (i) those which permitted normal expression, (ii) those which completely prevented expression, and (iii) those which permitted anaerobic expression of the operon but prevented any additional induction by nitrate. The nucleotide sequence was determined for a segment of the nar promoter region starting at one of the latter deletion end points and extending into the first structural gene of the operon. The position of the deletion end point relative to the translation start site for the first structural gene, narG, was defined by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. Deletions terminating 161 base pairs (bp) and approximately 200 bp upstream from the narG translation start site permitted anaerobic formation of nitrate reductase but interfered with the stimulation of nar operon expression by nitrate. A maximum size for the regulatory region was defined by two Tn5 insertions, which mapped approximately 550 bp 5' from the translation start site and did not interfere with the normal expression of nitrate reductase under anaerobic conditions with or without nitrate. We conclude that the nar operon 5' regulatory region is divided into two distinct regions: the 100 to 150 bp immediately 5' to the narG gene include a transcriptional start site and the signals necessary for anaerobic expression of the operon, and an adjacent region of 50 to 400 bp is required for the stimulation of operon expression by nitrate.