Methane formation by cell-free extracts of Methanobacillus omelianskii was studied. The extracts, prepared from crushed cells suspended in a neutral buffer, produced methane when hydrogen, carbon dioxide, coenzyme A (CoA), and adenosine triphosphate (ATP) were present. The reaction was optimal under a hydrogen atmosphere with 20% carbon dioxide. Methane formation was dependent on ATP and was stimulated by CoA. A concentration of 7.5 μmoles of ATP per ml effectively saturated the system. Pyruvate, serine, or O-phosphatase could substitute for ATP and carbon dioxide. Pyruvate was most effective at 0.04 m sodium pyruvate. Serine and O-phosphoserine elicited methane formation, while other amino acids did not. Ferredoxin stimulated methane formation but was unstable in the extracts. The role of ferredoxin was unclear, as it only stimulated methane formation when added to a reaction mixture containing pyruvate. The most active extracts from cells grown in high iron-containing medium were not stimulated by normal levels of ferredoxin, and at higher concentrations, ferredoxin had an inhibitory effect. The formation of methane in the presence of serine has interesting implications, as it may indicate that carbon atoms from serine or pyruvate are converted to methane. The ratio of pyruvate to methane formation varied from 20:1 to 100:1. The strictly anaerobic nature of methane formation has been a challenge in elucidating its biochemical mechanism. The experiments showed that active extracts could be prepared by conventional techniques if exposed to air for only short periods. However, the sensitivity to oxygen has prevented fractionation of the component enzymes. The study also found that ethanol, although a substrate for growth, was ineffective for methane formation when added to extracts of M. omelianskii. The role of hydrogen in methane formation was highlighted, as hydrogen was produced from ethanol by whole cells in the absence of carbon dioxide. The study provides insights into the biochemical mechanisms of methane formation by Methanobacillus omelianskii.Methane formation by cell-free extracts of Methanobacillus omelianskii was studied. The extracts, prepared from crushed cells suspended in a neutral buffer, produced methane when hydrogen, carbon dioxide, coenzyme A (CoA), and adenosine triphosphate (ATP) were present. The reaction was optimal under a hydrogen atmosphere with 20% carbon dioxide. Methane formation was dependent on ATP and was stimulated by CoA. A concentration of 7.5 μmoles of ATP per ml effectively saturated the system. Pyruvate, serine, or O-phosphatase could substitute for ATP and carbon dioxide. Pyruvate was most effective at 0.04 m sodium pyruvate. Serine and O-phosphoserine elicited methane formation, while other amino acids did not. Ferredoxin stimulated methane formation but was unstable in the extracts. The role of ferredoxin was unclear, as it only stimulated methane formation when added to a reaction mixture containing pyruvate. The most active extracts from cells grown in high iron-containing medium were not stimulated by normal levels of ferredoxin, and at higher concentrations, ferredoxin had an inhibitory effect. The formation of methane in the presence of serine has interesting implications, as it may indicate that carbon atoms from serine or pyruvate are converted to methane. The ratio of pyruvate to methane formation varied from 20:1 to 100:1. The strictly anaerobic nature of methane formation has been a challenge in elucidating its biochemical mechanism. The experiments showed that active extracts could be prepared by conventional techniques if exposed to air for only short periods. However, the sensitivity to oxygen has prevented fractionation of the component enzymes. The study also found that ethanol, although a substrate for growth, was ineffective for methane formation when added to extracts of M. omelianskii. The role of hydrogen in methane formation was highlighted, as hydrogen was produced from ethanol by whole cells in the absence of carbon dioxide. The study provides insights into the biochemical mechanisms of methane formation by Methanobacillus omelianskii.