Aging alters cardiac physiology and structure and enhances damage during ischemia and reperfusion. Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located among the myofibers, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Aging decreased the rate of oxidative phosphorylation using durohydroquinone, an electron donor to complex III, in IFM only. Complex III activity was decreased in IFM, but not SSM. Aging did not alter the content of catalytic centers of complex III (cytochromes b and c(1)and iron-sulfur protein). Complex III activity measured at physiologic ionic strength in IFM from aging hearts was decreased by 49% compared to IFM from adults, whereas activity measured at low ionic strength was unchanged, localizing the aging defect to the cytochrome c binding site of complex III. Subunits VIII and X of the cytochrome c binding site were present in complex III with the aging defect, indicating that loss of subunits did not occur. Study of aging damage to complex III will help clarify the contribution of altered electron transport in IFM to increased oxidant production during aging, formation of the aging cardiac phenotype, and the relationship of aging defects to increased damage following ischemia.