Legume nodule senescence is a poorly understood process involving a decrease in N(2) fixation and an increase in proteolytic activity. Some physiological changes during nodule aging have been reported, but scarce information is available at the subcellular level. Biochemical, immunological and proteomic approaches were used to provide insight into the effects of aging on the mitochondria and cytosol of nodule host cells. In the mitochondria, the oxidative modification of lipids and proteins was associated with a marked decline in glutathione, a reduced capacity to regenerate ascorbate, and upregulation of alternative oxidase and manganese superoxide dismutase. In the cytosol, there were consistent reductions in the protein concentrations of carbon metabolism enzymes, inhibition of protein synthesis and increase in serine proteinase activity, disorganization of cytoskeleton, and a sharp reduction of cytosolic proteins, but no detectable accumulation of oxidized molecules. We conclude that nodule mitochondria are an early target of oxidative modifications and a likely source of redox signals. Alternative oxidase and manganese superoxide dismutase may play important roles in controlling ROS concentrations and the redox state of mitochondria. The finding that specific methionine residues of a cytosolic glutamine synthetase isoform are sulfoxidized suggests a regulatory role of this enzyme in senescing nodules.