Glucagon and glucagon fragments from the carboxyl terminal end of the protein act at 0.01 to 0.1 mM concentrations on isolated rat liver mitochondria to sustain the rate of fixation of CO2 in the presence of pyruvate. The rate of decarboxylation of pyruvate is also increased by these substances. Similar effects are found with bacitracin, vanocomycin and cephalothin but not with any other of the proteins, peptides and antibiotics tested. The action of glucagon requires the presence of added magnesum ion. The addition of glucagon results in a better maintenance of adenosine triphosphate (ATP), and it leads to a greater degree of swelling of the mitochondria during the incubation. The effect of glucagon is partially mimicked by atractyloside, but it appears that glucagon is not exerting its effect by an atractyloside-like action. Added ATP obliterates the effect of added glucagon by sustaining fixation of CO2 However in incubations made in the presence of lower than usual levels of inorganic phosphate (2 mM vs. 8 mM) an effect of glucagon can be seen in the presence of ATP.