The kinetics of uptake of Ca(2+) by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients measured with or without added phosphate increase with the amount of Ca(2+) added up to about 24mum. Evidence is given that the effect is attributable to a combination of formation of chelates at low concentrations to act as Ca(2+) buffers, with co-transport of substrate to provide more respiratory fuel. The inhibitory effect of Mg(2+) depends on the Ca(2+) concentration, so with a constant [Mg(2+)] the low concentrations of Ca(2+) are most inhibited, and the rate coefficients are still more Ca(2+)-dependent. Ca(2+) uptake is slowed by local anaesthetics such as butacaine and dibucaine, and also by propranolol and palmitoyl-CoA. After an uptake, the release of Ca(2+) was investigated. The spontaneous release involves an initially slow and small appearance of free Ca(2+) and is followed by an auto-accelerated phase. The release is accompanied by a gradual decrease in internal ATP; it is initiated by palmitoyl-CoA (reversed by carnitine), by lysophosphatidylcholine, by Na(+) salts (reversed by oligomycin) and by K(+) salts added to a K(+)-free medium containing valinomycin. The process is probably a response to an increased energy load imposed on the mitochondria by the various conditions, which include the spontaneous action of phospholipase activated by traces of Ca(2+). The problem of how much mitochondrial activity is participating in normal heart Ca(2+) turnover is discussed, and experiments showing only 7-14% exchange of the mitochondrial Ca(2+) occurring in vivo in 10 or 20min are reported.