The effect of insulin (injected intraperitoneally) on the transport of Ca2+ by hapatic mitochondria from rats was investigated. Elevated concentrations of plasma insulin within the physiological range (10-100 μunits/ml) stimulate the initial rate of Ca2+ transport into mitochondria at 4°C by about 75% and prolong by approx. tenfold the time for which the mitochondria retain the accumulated Ca2+. The prolonged retention of Ca2+ is observed under the conditions where hypoglycaemia is significantly decreased by the simultaneous injection of glucose and insulin. A good correlation is observed between the effects on Ca2+ transport and the decrease in blood glucose concentration when the amount of insulin injected was varied. The effects of insulin on mitochondrial Ca2+ transport are apparent at about 30 min after the injection, and are inhibited by cycloheximide. There is little change in mitochondrial energy transduction after the administration of insulin. The results are briefly discussed in relation to the mechanism of Ca2+ transport across the inner mitochondrial membrane and the role of mitochondria in modifying intracellular Ca2+ concentrations with reference to the mechanism(s) by which insulin affects cellular metabolism.