Nitrate reductase, encoded by the nar operon in Escherichia coli, is produced only under anaerobic conditions and induced to its maximum level in the presence of nitrate. The anaerobic expression of the nar operon depends on the fnr gene product (Fnr), and the stimulation of anaerobic expression by nitrate requires the narL gene product (NarL). Distinct regulatory domains within the nar promoter are involved in these two responses. The specific locations of the sequences required for these two regulatory mechanisms were identified by analysis of a detailed set of deletions extending into the regulatory region of the nar operon from the 5' end. A region located around -55 base pairs (bp) from the transcriptional start site and immediately upstream from the presumed RNA polymerase binding site was required for the response to Fnr and anaerobic conditions. A base sequence no longer than 27 bp, located at about -200 bp, was essential for the stimulation by nitrate coupled with NarL. This NarL-specific sequence was equally effective if positioned 10 or 11 bp further upstream or downstream from its wild type position. However, it was ineffective if positioned 4, 6, or 14 bp or greater distances either upstream or downstream. Apparent autoregulation by active nitrate reductase occurred in all 5'-deletion constructions which retained the Fnr response, indicating that this regulatory phenomenon involves sequences located no further than -64 bp from the transcription start site.