Ca2+ exchange has been measured in a suspension of rat ventricular myocytes treated with digitonin or saponin to render the sarcolemma permeable to small molecules and ions. Two fractions of exchange were identified, one that was attributed to the mitochondrial component of the cell and the other to a non-mitochondrial fraction. Mitochondrial Ca2+ uptake was blocked by sodium azide and depended on respiratory substrates whereas non-mitochondrial uptake occurred independently of these molecules but was dependent on ATP and creatine phosphate. Non-mitochondrial Ca2+ uptake could be induced at a Ca2+ concentration below 1 microM and the initial rate increased with concentration up to 100 microM. Uptake could be reversed by sulmazole (a caffeine-like substance) and this reversal in turn inhibited by ryanodine. These properties suggest that the major locus for non-mitochondrial Ca2+ exchange is at the sarcoplasmic reticulum. Ca2+ exchange could be modulated by a number of agents, including carnosine, but was unaffected by others, including Na+, inositol trisphosphate and cyclic AMP. A kinetic model of the data is presented, which incorporates similar data of Ca2+ uptake into the mitochondrial fraction. The rates of Ca2+ exchange measured in these experiments suggest that these two components of the cell can reduce the sarcoplasmic Ca2+ concentration rapidly enough to account for the observed transient nature of the isometric twitch. Furthermore, it is suggested that both non-mitochondrial and mitochondrial fractions of the cell could significantly contribute to tension relaxation in rat cardiac muscle.