-Cadmium (Cd) is a toxic metal and an important environmental pollutant that can strongly affect mitochondrial function and bioenergetics in animals. We investigated the mechanisms of Cd action on mitochondrial function of a marine mollusk (the eastern oyster Crassostrea virginica) by performing a top-down control analysis of the three major mitochondrial subsystems (substrate oxidation, proton leak, and phosphorylation). Our results showed that the substrate oxidation and proton leak subsystems are the main targets for Cd toxicity in oyster mitochondria. Exposure to 12.5 M Cd strongly inhibited the substrate oxidation subsystem and stimulated the proton conductance across the inner mitochondrial membrane. Proton conductance was also elevated and substrate oxidation inhibited by Cd in the presence of a mitochondrially targeted antioxidant, MitoVitE, indicating that Cd effects on these subsystems were to a large extent ROS independent. Cd did not affect the kinetics of the phosphorylation system, indicating that it has negligible effects on F 1, FO ATP synthase and/or the adenine nucleotide transporter in oyster mitochondria. Cd exposure altered the patterns of control over mitochondrial respiration, increasing the degree of control conferred by the substrate oxidation subsystem, especially in resting (state 4) mitochondria. Taken together, these data suggest that Cd-induced decrease of mitochondrial efficiency and ATP production are predominantly driven by the high sensitivity of substrate oxidation and proton leak subsystems to this metal. cadmium; reactive oxygen species; proton leak; substrate oxidation; mitovite TOXIC METALS SUCH AS CADMIUM (Cd) are important contaminants in marine ecosystems. Most (ϳ97%) Cd in the biosphere is derived from human activities, although in some coastal areas Cd levels are naturally elevated due to the leaching of Cdenriched soils or deposition of Cd-hyperaccumulating diatoms (3,28,54). Cd is an extremely toxic metal with no known biological function in animals. Because of its rapid bioaccumulation and long residence time in the organisms, Cd represents an important health concern for humans and wildlife (54 -55).Because of its affinity to thiol groups and nitrogen-containing ligands, Cd can bind to a broad range of biological macromolecules (such as proteins and DNA), damaging their structure and interfering with their functions (5). Mitochondria are key intracellular targets for Cd due to their ability to accumulate Cd and because of the sensitivity of mitochondrial enzymes to Cd-induced damage (14,48,56,73). Because of the central role of mitochondria in critical cellular processes such as bioenergetics, redox signaling, and cell death, Cdinduced mitochondrial damage has long-ranging consequences for cellular function, energy homeostasis, and whole-organism performance and survival. Previous studies have shown that Cd suppresses respiration and leads to the partial uncoupling, as well as elevated rates of reactive oxygen species (ROS) production in animal and plant mitochondr...