Summary: The effects of adrenaline on kinetically-distinct compartments of exchangeable calcium in the spontaneously-beating perfused rat heart were investigated under steady-state conditions using a 45Ca2+ outflow technique together with a nonlinear least-squares curve fitting procedure and 51Cr-EDTA to monitor the loss of freely-diffusible Ca2+ from the extracellular space. In addition to Ca2+ distributed in the vascular (compartment 1) and interstitial (compartment 2) spaces, the minimum number of kinetically-distinct compartments of cellular exchangeable Ca2+ (which include Ca2+ bound to extracellular sites on the sarcolemma) required to fit the data (compartments 3,4 and 5) was three. A system in which cellular compartment 3 is linked to the vascular space and compartments 4 and 5 are linked to the interstitial space was consistent with the data. For hearts perfused under control conditions, the fractional transfer rates for Ca2+ outflow from cellular compartments 3, 4 and 5 were 0.70, 0.11 and 0.017 min-1, respectively. Adrenaline increased the quantity of exchangeable Ca2+ in compartment 5 and decreased that present in the compartment 4. Evidence that compartment 5 includes mitochondrial exchangeable Ca2+ is discussed. It is concluded that one of the actions of adrenaline on the heart is to increase the quantity of exchangeable Ca2+ in the mitochondria.