Methanospirillum hungatei strains GP1 and JF1 when exposed to the adenosinetriphosphatase inhibitor N,N′-dicyclohexylcarbodiimide experienced a marked decline in growth rates, methane synthesis activities, and intracellular ATP concentrations. Although growth was inhibited, the intracellular ATP concentrations of all other methanogens tested were little affected by high concentrations of dicyclohexylcarbodiimide (500 μM). Thus, for studies of ATP synthesis, or for ATP depletion in whole cell suspensions of methanogens, the use of dicyclohexylcarbodiimide appears limited to M. hungatei.