The widespread occurrence and diversity of ammonia oxidizing Archaea suggests their contribution to the nitrogen cycle is of global significance. Their distribution appeared limited to low- and moderatetemperature environments until the recent finding of a diagnostic membrane lipid, crenarchaeol, in terrestrial hot springs. We report here the cultivation of a thermophilic nitrifier (‘Candidatus Nitrosocaldus yellowstonii’), an autotrophic crenarchaeote growing up to 74°C by aerobic ammonia oxidation. The major core lipid of this archaeon growing at 72°C is crenarchaeol, providing the first direct evidence for its synthesis by a thermophile. These findings greatly extend the upper temperature limit of nitrification and document that the capacity for ammonia oxidation is broadly distributed among the Crenarchaeota.