The effects of glucagon on cellular calcium distribution in cardiac muscle were studied using an isolated, spontaneously-beating, perfused rat heart preparation and a 45Ca outflow exchange technique. The rate of loss of 45Ca from the cellular space (intracellular calcium and calcium bound to extracellular sites) was determined, using [14C]sucrose to estimate the rate of loss of 45Ca from the extracellular space. The data were analysed by an iterative non-linear, least squares curve-fitting procedure. The simplest system which was found to be consistent with 45Ca outflow exchange data obtained for control and glucagon-treated hearts is one in which three kineticaliy-distinct compartments of exchangeable calcium are associated with the cellular space. For control hearts, the fractional transfer rates (rate constants) for the outflow of calcium from compartments 1-3 were 0.61, 0.14 and 0.022min-1, respectively. Measurement of the amounts of 45Ca present in subcellular fractions of perfused hearts indicated that compartment 3 represents calcium present in intracellular stores, including mitochondria and possibly the sarcoplasmic reticulum. The predominant effects of glucagon were to increase the fractional transfer rate for calcium outflow from compartment 1, and the quantity of exchangeable calcium in compartment 3.