The gram-negative anaerobe Oxalobacter formigenes, grows on oxalate as the principal carbon and energy source, but a small amount of acetate is also required for growth. Experiments were conducted to determine the distribution and the position of label in cellular amino acids from cells grown on [13C]oxalate, [13C]acetate (1-13C, 2-13C, and U-13C), and 13CCO3. The labeling pattern (determined with NMR spectroscopy) of amino acids was consistent with their formation through common biosynthetic pathways. The majority of the carbons in the amino acids that are usually derived from pyruvate, oxaloacetate, α-ketoglutarate, 3-phosphoglycerate, and carbon in the aromatic amino acids were labeled by oxalate. Carbon from 13CO3 was assimilated primarily into amino acids expected to be derived from oxaloacetate and α-ketoglutarate. Approximately 60% of the acetate that was assimilated into amino acids was incorporated as a C2 unit into proline, arginine, glutamate, and leucine. The pattern of labeling from acetate in glutamate, arginine, and proline was consistent with acetate incorporation via citrate (si)-synthase and subsequent formation of α-ketoglutarate via the first third of the tricarboxylic acid pathway. Acetate was also assimilated into amino acids derived from pyruvate and oxaloacetate, but results indicated that this incorporation was as single carbon atoms. Based on these findings, cell-free extracts were assayed for several key biosynthetic enzymes. Enzymatic activities found included glutamate dehydrogenase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase. These findings are consistent with proposed biosynthetic mechanisms.