The rat mitochondrial proteome was analyzed using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and proteins altered by age or caloric restriction (CR) were identified using mass spectrometry. Of 2061 mitochondrial proteins analyzed in the three tissues, a significant change with age occurred in 25 liver proteins (19 increased, 6 decreased), 3 heart proteins (1 increased, 2 decreased), and 5 skeletal muscle proteins (all increased). CR prevented the age-related change in the level of one liver mitochondrial protein, altered the levels of four proteins (one increased, three decreased) from heart, and one protein (decreased) from skeletal muscle. Identification of the proteins that changed with age or CR revealed that they were varied among the three tissues, that is, not one mitochondrial protein was changed, in common, by age or CR in any tissue studied. Thus, the effect of age on the mitochondrial proteome appears to be tissue-specific, and CR has a minor effect on age-related protein changes.