Extracts of acetone powder of rat liver mitochondria, as well as the intact liver mitochondria, catalyzed the synthesis of 2 molecules of glycine from 1 molecule each of L-serine, bicarbonate and ammonia. The β-carbon of serine and bicarbonate carbon were incorporated specifically into the α-carbon and the carboxyl carbon of glycine, respectively. Ammonia seemed to be the direct and specific amino donor in the glycine synthesis. The reaction required tetrahydrofolate and reducing agents such as dithio-threitol or NADH, but the reaction did not require the exogenous supply of ATP or GTP.