This project is designed to assess the physiological importance of calcium ions in the regulation of energy metabolism. We have exposed isolated rat cardiac myocytes to a variety of agents and conditions expected to alter cytosolic free calcium ion concentration and have measured the rate of 02-uptake by the cell suspensions, as well as the cellular content of NADH. Further, we have estimated cytosolic calcium concentration using fluorescent chelating agents, in parallel experiments performed under identical conditions. In each case, there was a good correlation between the magnitude of the increase in calcium concentration and the degree of stimulation of 02-uptake. Attempts were made to assess the quantitative significance of direct activation of respiration by calcium ions at the level of mitochondrial dehydrogenases versus an indirect mechanism involving increased ADP generation. Ruthenium red, which blocks the former process but not the latter, gave a small decrease in rates of 02-uptake. However, activation of oxidative phosphorylation by ADP was a predominant mechanism, based on the lowered mitochondrial content of NADH which was observed to occur in response to calcium mobilization.