Exposure of isolated hepatocytes to glucagon for 45 min caused a 2.5-fold increase in the time (Ca2+ retention time) for which mitochondria subsequently isolated from the cells retained a load of exogenous Ca2+ before its spontaneous release. Half maximal effect of glucagon was observed at a concentration of 0.6 nM. An increase in the Ca2+ retention time was observed after 30 but not 15 min exposure of cells to the hormones. Incubation of hepatocytes with dexamethasone, epinephrine, vasopressin, dibutyryl cyclic AMP or 8-bromo cyclic GMP also induced an increase in mitochondrial Ca2+ retention time. The effect of glucagon was associated with an increase in cellular cyclic AMP and was inhibited by puromycin, cycloheximide and cordycepin, but not by actinomycin D or chloramphenicol. Puromycin caused only a small inhibition of the stimulation by glucagon of mitochondrial pyruvate carboxylation. It is concluded that the effects of glucagon on mitochondrial Ca2+ retention require nuclear DNA-directed protein synthesis and differ, in this respect, from the rapid-onset effects of the hormone on other mitochondrial properties, including pyruvate carboxylation.