The concentration of cholesterol in the plasma of rats maintained on a diet enriched by the addition of cholesterol (1.8%, w w) and olive oil (14% w w) for periods of 7 or 16 weeks increased 1.7-fold compared to that in rats maintained on a control diet, or one enriched with olive oil alone. Liver mitochondria isolated from rats maintained on a diet enriched with cholesterol and olive oil, or with cholesterol alone, exhibited increases of two- to threefold in cholesterol concentration and the time for which a given load of exogenous calcium is retained when compared with mitochondria isolated from control rats, or rats maintained on a diet enriched with olive oil alone. No change in the rates of respiration or the concentration of endogenous inorganic phosphate was observed. Heart mitochondria isolated from rats maintained on a diet enriched with cholesterol and olive oil showed no increase in cholesterol concentration or the time for which calcium is retained compared to control mitochondria. An increase of 20% in the ratio of the ADP-stimulated: ADP-depleted rates of respiration was observed in mitochondria isolated from the hearts of rats maintained on the enriched diet compared with mitochondria isolated from rats maintained on a diet of olive oil alone. It is proposed that (a) the increased calcium retention exhibited by liver mitochondria enriched with cholesterol results from changes in the membrane induced by the presence of high concentrations of this compound and (b) heart mitochondria or cells are more resistant than those of the liver to infiltration by cholesterol.