To identify possible relationships between the loss of calcium homeostasis in brain associated with aging and alterations in the function of key calcium regulatory proteins, we have purified calmodulin (CaM) from the brains of Fischer 344 rats of different ages and have assessed age-related alterations in (i) the secondary and tertiary structure of CaM and (ii) the ability of CaM to activate one of its target proteins, the plasma membrane (PM) Ca-ATPase. There is a progressive, age-dependent reduction in the ability of CaM to activate the PM-Ca-ATPase, which correlates with the oxidative modification of multiple methionines to their corresponding methionine sulfoxides. No other detectable age-related posttranslational modifications occur in the primary sequence of CaM, suggesting that the reduced ability of CaM to activate the PM-Ca-ATPase is the result of methionine oxidation. Corresponding age-related changes in the secondary and tertiary structure of CaM occur, resulting in alterations in the relative mobility of CaM on polyacrylamide gels, differences in the intrinsic fluorescence intensity and solvent accessibility of Tyr99 and Tyr138, and a reduction in the average alpha-helical content of CaM at 20 degreesC. Shifts in the calcium- and CaM-dependent activation of the PM-Ca-ATPase are observed for CaM isolated from senescent brain, which respectively requires larger concentrations of either calcium or CaM to activate the PM-Ca-ATPase. The observation that the oxidative modification of CaM during normal biological aging results in a reduced calcium sensitivity of the PM-Ca-ATPase, a lower affinity between CaM and the PM-Ca-ATPase, and the reduction in the maximal velocity of the PM-Ca-ATPase is consistent with earlier results that indicate the calcium handling capacity of a range of tissues including brain, heart, and erythrocytes isolated from aged animals declines, resulting in both longer calcium transients and elevated basal levels of intracellular calcium. Thus, the oxidative modification of selected methionines in CaM may explain aspects of the loss of calcium homeostasis associated with the aging process.