FNR is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli. The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions. Repression is not simply due to occlusion of the promoter (-35 and -10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at -50.5 and -94.5 in the ndh promoter region without preventing RNA polymerase binding. However, contact between RNA polymerase and the -132 to -62 region of the non-coding strand of ndh DNA, and RNA polymerase-mediated open complex formation, were prevented by bound FNR. The upstream FNR-binding site (-94.5) was needed for efficient FNR-dependent repression of ndh transcription in vitro, and also for repression of an ndh-lacZ fusion in vivo. Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the -35 region, which prevents essential RNA polymerase-DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction. Expression of the ndh-lacZ fusion in an fnr deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (M(r) 12,000 and 35,000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis. These putative amino acid-responsive activators may thus offset FNR-mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD+/NADH ratio during growth in rich media.