1. Mitochondria isolated from rats treated with glucagon for 60 min or livers perfused in the presence of glucagon for 10min exhibited lower rates of 45Ca2+ exchange than did control mitochondria when this was measured under steady-state conditions in the presence of Mg2+, ATP, P, and 0.13 pM- or 0.16pgM-free Ca2+ at pH7.4 and at 25C or 37C. Under these conditions no significant difference in the rates of Ruthenium Red-induced 45Ca2+ efflux was observed. These results contrast with earlier work in which mitochondria isolated from glucagon-treated livers were shown to exhibit faster rates of Ca2+ uptake [Yamazaki (1975) J. Biol. Chem. 250, 7924-79301 and slower rates of spontaneous Ca2+ efflux [Hughes & Barritt (1978) Biochem. J. 176, 295-3041 when these parameters were measured under different incubation conditions, including supra-physiological concentrations of free Ca2+ and the absence of added Mg2+ and ATP. 2. Perfusion of livers with glucagon before the addition of adrenaline or the Ca2+-selective ionophore A23187, to release Ca2+ from intracellular stores, decreased the amount of Ca2+ released by these agents. 3. Incubation of isolated hepatocytes in the presence of glucagon at 1.3mm extracellular Ca2+ induced a small decrease in the plateau of the 45Ca2+-exchange curve obtained under steady-state conditions. 4. It is concluded that the actions of glucagon on liver mitochondrial Ca2+ transporters lead to a decrease, rather than an increase, in mitochondrial Ca2+ stores in the intact cell.