The nucleotide sequence of the region probably responsible for regulation of pepN expression and of the region encoding the amino-terminal part of aminopeptidase N, has been determined. The transcription start site was identified by S1 nuclease mapping. All features of the promoter are those of a weak promoter and no obvious structure responsible for regulation was identified, although a possible Pho box is located 63 base pairs upstream from the Pribnow box. The reading frame was unambiguously determined by purifying the protein and by sequencing the first 21 NH2-terminal residues. The NH2-terminal region of aminopeptidase N does not contain any fragment resembling signal sequence and the protein is not produced in a precursor form. A divergent promoter, which might be that of pncB, encoding the nicotinic acid phosphoribosyltransferase (P. Terpstra, personal communication), has also been identified, which allows the assignment of the gene organization in this chromosomal region as ompF asnS pncB pepN.