Several lines of evidence, including the reported ability of calcium channel blockers to prevent atherogenesis in cholesterol-fed rabbits, suggest that calcium mediates one or more of the pathologic changes in atherosclerosis. Moreover, it has long been known that calcium accumulates in atherosclerotic blood vessels. To test the hypothesis that a substantial fraction of this accumulated calcium is intracellular and to identify possible causes of this accumulation, calcium fluxes and contents were determined in aortic segments from cholesterol-fed rabbits and age-matched controls. A new method, based on 45Ca efflux experiments and computer-assisted kinetic analysis, was used to measure intracellular and extracellular calcium contents (nmol calcium/g wet wt tissue) and fluxes. Total intracellular calcium increased from 269 +/- 11.6 to 1,300 +/- 352 nmol/g in cholesterol-fed animals compared with controls (p less than 0.01). This change was sufficient to account for the observed increase in total tissue calcium from 4,190 +/- 211 to 5,240 +/- 477 nmol/g (p less than 0.05). Thus, the fraction of tissue calcium that is intracellular increased significantly from 0.065 +/- 0.006 to 0.223 +/- 0.048 (p less than 0.01) in experimental atherosclerosis. In addition, the data were quantitatively consistent with the hypothesis that these changes are brought about by a 4.8-fold increase in the plasma membrane calcium permeability of aortic smooth muscle cells. These results provide evidence that increased intracellular calcium is a possible mediator of cholesterol-induced atherogenesis.