A tritium exchange assay and a sensitive gas chromatographic technique were used to demonstrate that hydrogenase was active and that hydrogen was produced by Methanosarcina barkeri strain MS grown on acetate. Both methane and hydrogen production rates were dependent on the concentration of acetate in the medium. H2 was produced at 0.5–2% of the rate of CH4 formation. Chloroform and potassium cyanide, inhibitors of methanogenesis from acetate, inhibited H2 production but not hydrogenase activity. The addition of hydrogen gas to cell suspensions did not inhibit CH4 or carbon dioxide production from the methyl group of acetate. H2 production appears to be linked to several intracellular redox processes which follow the cleavage of acetate.