Spirochetes from termite hindguts and freshwater sediments possessed homologs of a nitrogenase gene ( nifH ) and exhibited nitrogenase activity, a previously unrecognized metabolic capability in spirochetes. Fixation of 15-dinitrogen was demonstrated with termite gut Treponema ZAS-9 and free-living Spirochaeta aurantia . Homologs of nifH were also present in human oral and bovine ruminal treponemes. Results implicate spirochetes in the nitrogen nutrition of termites, whose food is typically low in nitrogen, and in global nitrogen cycling. These results also proffer spirochetes as a likely origin of certain nifH s observed in termite guts and other environments that were not previously attributable to known microbes.