Mitochondria extracted from the hearts of Wistar rats aged 6 and 24 months showed similar values for the respiratory control index (RCI), state 3 oxygen consumption (QO2) and ADP/O measured using glutamate or succinate as substrates; with the exception that the QO2 of the aged rats was lower than that of the young rats in the presence of glutamate. The consumption of O2 induced by 2-oxoglutarate and ADP was similar in both age groups. Concentrations of external free Ca2+ ranging from 0.2 to 0.8 microM produced an increase in O2 consumption and ATP formation in the young mitochondria, with a maximum effect at 0.2 microM external free Ca2+. Little or no change in O2 consumption and ATP formation was evident in aged mitochondria following incubations in concentrations of external free Ca2+ ranging from 0.2 to 0.8 microM. The continuous rate of formation of ATP, measured in the presence of 0.2 microM external free Ca2+ using a luminescence method, confirmed the previous results. This study indicates that the cardiac mitochondrial phosphorylating system of aged rats is poorly sensitive to variations in external free calcium.