The differentiation of chloroplasts into chromoplasts involves a series of biochemical changes that culminate with the intense accumulation of long chain chromophore carotenoids such as lycopene, rhodoxanthin, astaxanthin, anhydroeschsoltzxanthin, capsanthin, and capsorubin. The signal pathways mediating these transformations are unknown. Chromoplast carotenoids are known to accumulate in green tissues experiencing stress conditions, and studies indicate that they provide efficient protection against oxidative stress. We tested the role of reactive oxygen species (ROS) as regulators of chromoplast carotenoid biosynthesis in vivo. The addition of ROS progenitors, such as menadione, tert-butylhydroperoxide, or paraquat and prooxidants such as diamide or buthionine sulfoximine to green pericarp discs of pepper fruits rapidly and dramatically induce the simultaneous expression of multiple carotenogenic gene mRNAS that give rise to capsanthin. Similarly, down-regulation of catalase by amitrole induces expression of carotenogenic gene mRNAs leading to the synthesis of capsanthin in excised green pericarp discs. ROS signals from plastids and mitochondria also contribute significantly to this process. Analysis of the capsanthin-capsorubin synthase promoter in combination with a -glucuronidase reporter gene reveals strong activation in transformed pepper protoplasts challenged with the above ROS. Collectively these data demonstrate that ROS act as a novel class of second messengers that mediate intense carotenoid synthesis during chromoplast differentiation.Plastids are plant organelles whose diverse functions include photosynthesis, gravity perception, and biogenesis of microand macromolecules. These functions do not occur in all plastids but are associated with structurally distinct plastid types. Plastid differentiation is a highly coordinated process involving programmed, multi-phase events that are transduced by a variety of stimuli. These transducers activate numerous morphological and biochemical changes that ultimately affect plastid compartmentalization. This is particularly evident in chloroplasts undergoing the transformation to chromoplasts, an event characterized by the synthesis and accumulation of carotenoids into unique plastid substructures. These changes are accompanied by the yellow to red color shift in flowers, fruits, and roots of certain plants (1, 2). Although the molecular events controlling these alterations are largely unknown, emerging evidence suggests that reactive oxygen species (ROS) 1 may play a vital role in this process (3). In this paper we present data showing how oxidative stress affects carotenogenesis in plants.Data from several model systems indicate that ROS operate at the molecular level. Homeostatic variations in ROS levels are known to activate normal and pathological events during animal cell development (4, 5).