Expression of the narK gene of Escherichia coli, like the narGHJI operon, is positively regulated by two trans-acting factors: Fnr, which is activated by anaerobic conditions, and NarL, which is activated by the conditions, and NarL, which is activated by the presence of nitrate. Unlike the narGHJI operon, the 5' untranslated region of the narK gene contains two putative Fnr-binding-site sequences and two putative NarL-binding-site sequences. To define the role of these putative cis-acting regions, transcription start sites were identified and the effects of promoter region modifications on transcription were determined. Primer extension analysis identified several transcripts for the narK gene expressed from plasmids. Expression from the major promoter, P1, was induced by anaerobic growth conditions and further elevated in the presence of nitrate, while that from a weaker promoter, P2, appeared to be constitutive. The position of the major transcription start site placed one of the putative Fnr-binding sites (Fnr1 box) and one of the NarL-binding sites (NarL2 box) at positions analogous to those previously established for the narGHJI operon promoter region, while the other two binding sites were located in the non-homologous 150 bp sequence which separates the Fnr1 and NarL2 boxes. Based on the effects of selective 5' deletions and site-directed modifications, Fnr-dependent expression was dependent only on the Fnr1 box and nitrate stimulation was dependent on the presence of the NarL2 box. In the absence of the NarL2 box, the NarL1 box did not promote stimulation by nitrate. The Fnr2 box was not required for anaerobic induction of expression but its modification appeared to reduce the level of stimulation by nitrate.