One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term (14)CH(3)OH or (14)CO(2) incorporation studies during growth and by cell suspensions. (14)CH(3)OH and (14)CO(2) were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H(2)-CO(2)-methanol. (14)CH(3)OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas (14)CO(2) was selectively incorporated into the C(1) moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, (14)CH(4) and [3-(14)C]alanine synthesized from (14)CH(3)OH during growth shared a common specific activity distinct from that of CO(2) or methanol. Cell suspensions synthesized acetate and alanine from (14)CO(2). The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of (14)CH(3)OH or (14)CO(2) fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from (14)CH(3)OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from (14)CO ( approximately 1 nmol/min per mg of protein) and an isotopic exchange between CO(2) or CO and the C-1 of pyruvate. Acetate synthesis from (14)CO was stimulated by methyl B(12) but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (>6 mumol/min per mg of protein) and acetate kinase (>0.14 mumol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.