The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) and the NifA homologue, AQ_218. Purified A. aeolicus sigma(N) bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFDeltaHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.