Calcium is an important regulator of mitochondrial function. Since there can be tight coupling between inositol 1,4, 5-trisphosphate-sensitive Ca(2+) release and elevation of mitochondrial calcium concentration, we have investigated whether a similar relationship exists between the release of Ca(2+) from the ryanodine receptor and the elevation of mitochondrial Ca(2+). Perfusion of permeabilized A10 cells with inositol 1,4, 5-trisphosphate resulted in a large transient elevation of mitochondrial Ca(2+) to about 8 microm. The response was inhibited by heparin but not ryanodine. Perfusion of the cells with Ca(2+) buffers in excess of 1 microm leads to large increases in mitochondrial Ca(2+) that are much greater than the perfused Ca(2+). These increases, which average around 10 microm, are enhanced by caffeine and inhibited by ryanodine and depletion of the intracellular stores with either orthovanadate or thapsigargin. We conclude that Ca(2+)-induced Ca(2+) release at the ryanodine receptor generates microdomains of elevated Ca(2+) that are sensed by adjacent mitochondria. In addition to ryanodine-sensitive stores acting as a source of Ca(2+), Ca(2+)-induced Ca(2+) release is required to generate efficient elevation of mitochondrial Ca(2+).