We have previously proposed that hypercholesterolemic LDL receptor knockout (K/o) mice mitochondria possess a lower antioxidant capacity due to a large consumption of reducing equivalents from NADPH to sustain high rates of lipogenesis (FASEB J. 2005; 19: 278-280). In this work, the K/o mice oxidative stress was further characterized by showing a lower mitochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to control mice. No differences in the activity of the antioxidant enzyme system glutathione reductase/peroxidase were observed. Exogenous catalase prevented the spontaneous oxidation of endogenous NADPH in rotenone poisoned mitochondria isolated from k/o mice, indicating that this oxidation is mediated by mitochondria generated H₂O₂. The higher rate of ROS production in K/o mitochondria was also prevented by the presence of exogenous isocitrate which maintains NADP fully reduced. Our hypothesis that high rates of lipogenesis decreases NADPH/NADP⁺ ratio due to a decreased content of NADP-linked substrates is supported by two distinct observations: i) oxygen consumption supported by endogenous NADP-linked substrates was lower in K/o mice than in control mitochondria, but was similar in the presence of exogenous isocitrate; ii) when K/o mice were treated with drinking water containing 55 mM sodium citrate/67 mM citric acid during one week, both the rate of oxygen consumption supported by NADP-linked substrates and the mitochondrial capacity to sustain reduced NADPH were partially restored. In conclusion, the data demonstrate that the mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate.