Oxidative stress can occur in different parts of plant cells. We employed two oxidants that induce reactive oxygen species (ROS) in different intracellular compartments: methyl viologen (MV, in chloroplasts) and menadione (MD, in mitochondria). The responses of pea (Pisum sativum) leaf discs to MV or MD after 4-h incubation in dark or moderate (300 μE m-2 s-1) or high light (1200 μE m-2 s-1) were examined. Marked increase in ROS levels was observed, irrespective of compartment targeted. The levels of proline, a compatible solute, increased markedly much more than that of ascorbate or glutathione during oxidative/photo-oxidative stress, emphasizing the importance of proline. Further, the activities and transcripts of enzymes involved in biosynthesis or oxidation of proline were studied. An upregulation of biosynthesis and downregulation of oxidation was the basis of proline accumulation. Pyrroline-5-carboxylate synthetase (P5CS, involved in biosynthesis) and proline dehydrogenase (PDH, involved in oxidation) were the key enzymes regulated under oxidative stress. Since these two enzymes-P5CS and PDH-are located in chloroplasts and mitochondria, respectively, we suggest that proline metabolism can help to mediate inter-organelle interactions and achieve redox homeostasis under photo-oxidative stress.