Oxalobacterformigenes is the only well-documented oxalate-degrading bacterium isolated from the gastrointestinal tract of animals. The production of ATP by Oxalobacter formigenes is centered around oxalate metabolism and oxalate is required for growth. A small amount of acetate (0.5 mM) is also required. Oxalate is decarboxylated to formate plus CO2 in nearly equimolar amounts. Experiments were conducted to determine which potential carbon sources (oxalate, acetate, formate, CO2) were assimilated by Oxalobacter formigenes and which metabolic pathways were operative in carbon assimilation. Measurements of the specific activities of total cell carbon after growth with different 14C-labeled precursors indicated that at least 54% of the total cell carbon was derived from oxalate and at least 7% was derived from acetate. Carbonate was also assimilated, but formate was not a significant source of cell carbon. Labeling patterns in amino acids from cells grown in [14C]oxalate or 14CO3 were different; however, in both cases 14C was widely distributed into most cellular amino acids. Carbon from [14C]acetate was less widely distributed and detected mainly in those amino acids known to be derived from α-ketoglutarate, oxaloacetate, and pyruvate. Cell-free extracts contained citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities. The labeling observed in amino acids derived from acetate is in agreement with the function of these enzymes in biosynthesis and indicates that the majority of acetate carbon entered into amino acid biosynthesis via well-known pathways.