Acetylcholine (ACh) is known generally as the neurotransmitter in the mammalian central and peripheral cholinergic nervous systems. However, ACh is also widely expressed in non-neuronal animal tissues and in plants, fungi and bacteria, where it is likely involved in the transport of water, electrolytes and nutrients, and in modulating various other cell functions. We have investigated the expression of ACh and ACh-synthesizing activity in various strains of Archaea, which are situated between Bacteria and Eucarya in the universal phylogenetic tree. Using a sensitive and specific radioimmunoassay, differing levels of ACh were detected in the Hyperthermophiles Thermococcus kodakaraensis KOD1, Sulfolobus tokodaii strain 7 and Pyrobaculum calidifontis VA1; the Methanogens Methanothermobacter thermautotrophicus deltaH and Methanosarcina barkeri; and the Halophiles Halobacterium sp. NRC-1 and Haloferax volcanii. T. kodakaraensis KOD1 expressed the highest levels of ACh among the Archaea tested; moreover, the substance expressed was verified to be ACh using high-performance liquid chromatography with electrochemical detection. Varying degrees of ACh-synthesizing activity were also identified in all of the strains, and the activity of bromoACh-sensitive choline acetyltransferase, an enzyme responsible for ACh synthesis in the nervous system, was detected in T. kodakaraensis KOD1. Our findings demonstrate that ACh and ACh-synthesizing activity are both expressed in evolutionally old Archaea. In the context of the recent discovery of non-neuronal ACh in bacteria, fungi, plants and animals, these findings support the notion that ACh has been expressed in organisms from the origin of life on the earth, functioning as a local mediator as well as a neurotransmitter.