The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor ςN (ς54, RpoN) and five potential ςN-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 ςN-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 ςN and the NifA homologue, AQ_218. Purified A. aeolicus ςN 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 ςN-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus ςN supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFΔHTH 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 ςN enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and ςN have coevolved.