Oxidants can reversibly increase the permeability of endothelium to ions and macromolecules. Oxidants also deplete ATP in cultured endothelial cells. We asked if oxidant-mediated ATP depletion, alone, accounted for the effects of oxidants on endothelial permeability to macromolecules. When porcine pulmonary artery endothelial cells were exposed to 2.5 mmol/LH2O2, ATP was depleted to 31.7% +/- 1.8% of control within 15 minutes and was reduced to 23.1% +/- 2.0% of control after 30 minutes. To determine if this magnitude of ATP depletion could account for the oxidant-induced increase in endothelial permeability to macromolecules, we measured ATP in endothelial cells exposed to metabolic inhibitors of ATP production. We then measured the effects of these metabolic inhibitors on endothelial monolayer permeability to macromolecules. ATP levels were reduced to 44% +/- 4% of control by 12 mmol/L deoxyglucose (DOG) in the absence of glucose and to 2% +/- 1.3% of control by DOG with 25 nmol/L antimycin A in the absence of glucose. Reduction of endothelial cell ATP to these levels with the metabolic inhibitors did not alter the flux of albumin or dextran across the endothelial monolayers. Thus ATP depletion, by itself, does not explain oxidant-induced changes in endothelial permeability to macromolecules.