In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, approximately 20 mmHg) without glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by complex III inhibitor: antimycin A (24 +/- 3%, P < 0.0001), or stigmatellin (31 +/- 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 +/- 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.