We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24+/-6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N(epsilon)-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Deltapsi), ADP/O ratio, and the content of coenzyme Q(9), cytochromes b and a+a(3). Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=-0.56) ATP/ADP ratios, and Deltapsi (C=-0.11). We conclude that although HFD induces accumulation of LCAC and N(epsilon)-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.