In cardiac tissue two mitochondria subpopulations, the subsarcolemmal and the intermyofibrillar mitochondria, present different functional emphasis, although limited information exists about the underlying molecular mechanisms. Our study evidenced higher OXPHOS activity of intermyofibrillar compared to subsarcolemmal mitochondria, paralleled by distinct membrane proteins susceptibility to oxidative damage and not to quantitative differences of OXPHOS composition. Indeed, subsarcolemmal subunits of respiratory chain complexes were more prone to carbonylation while intermyofibrillar mitochondria were more susceptible to nitration. Among membrane protein targets to posttranslational modifications, ATP synthase subunits alpha and beta were notoriously more carbonylated in both subpopulations, although more intensely in subsarcolemmal mitochondria. Our data highlight a localization dependence of cardiac mitochondria OXPHOS activity and susceptibility to posttranslational modifications.