Hyperglycemic challenge to bovine aortic endothelial cells (BAECs) increases oxidant formation and cell damage that are abolished by MnSOD overexpression, implying mitochondrial superoxide (O(2)(.-)) as a central mediator. However, mitochondrial O(2)(.-) and its steady-state concentrations have not been measured directly yet. Therefore, we aimed to detect and quantify O(2)(.-) through different techniques, along with the oxidants derived from it. Mitochondrial aconitase, a sensitive target of O(2)(.-), was inactivated 60% in BAECs incubated in 30 mM glucose (hyperglycemic condition) with respect to cells incubated in 5 mM glucose (normoglycemic condition). Under hyperglycemic conditions, increased oxidation of the mitochondrially targeted hydroethidine derivative (MitoSOX) to hydroxyethidium, the product of the reaction with O(2)(.-), could be specifically detected. An 8.8-fold increase in mitochondrial O(2)(.-) steady-state concentration (to 250 pM) and formation rate (to 6 microM/s) was estimated. Superoxide formation increased the intracellular concentration of both hydrogen peroxide, measured as 3-amino-2,4,5-triazole-mediated inactivation of catalase, and nitric oxide-derived oxidants (i.e., peroxynitrite), evidenced by immunochemical detection of 3-nitrotyrosine. Oxidant formation was further evaluated by chloromethyl dichlorodihydrofluorescein (CM-H(2)DCF) oxidation. Exposure to hyperglycemic conditions triggered the oxidation of CM-H(2)DCF and was significantly reduced by pharmacological agents that lower the mitochondrial membrane potential, inhibit electron transport (i.e., myxothiazol), and scavenge mitochondrial oxidants (i.e., MitoQ). In BAECs devoid of mitochondria (rho(0) cells), hyperglycemic conditions did not increase CM-H(2)DCF oxidation. Mitochondrial O(2)(.-) formation in hyperglycemic conditions was associated with increased glucose metabolization in the Krebs cycle and hyperpolarization of the mitochondrial membrane.