Reactive oxygen species (ROS) are implicated in coronary collateral growth. Accordingly, we evaluated the requirement for ROS in endothelial cell (HCAEC) tube formation, coronary collateral development, and in redox sensitive activation of p38 MAP kinase. The flavin-containing oxidase inhibitor, DPI (10 µM) or the superoxide dismutase (SOD) inhibitor, DETC (10 mM) blocked VEGF-induced (50 ng/ml) HCAEC tube formation in Matrigel (p<0.05, n=10). DPI and DETC also inhibitied coronary collateral development in a rat model of episodic ischemia. Each day of the protocol, DPI (0.2 mg/kg/day), or DETC (1 g/kg/day) were given by IP injection, or the NAD(P)H oxidase inhibitor, apocynin (0.25 mg/ml) was given in drinking water. Collateral-dependent flow (radioactive microspheres) was expressed as a ratio of flows between the normal and the ischemic zone. In sham animals, collateral flow in the ischemic zone was 18±6% of the normal zone flow; whereas in the RI group collateral flow in the ischemic zone was 83±5% of that in the normal zone. Blockade of ROS (superoxide and H₂O₂) production by DPI prevented the increase in collateral flow after RI (25±4% of that in the normal zone). Nearly identical results were obtained with apocynin following RI (32±7% of that in the normal zone). Increasing superoxide levels and decreasing H₂O₂ levels by SOD inhibition (DETC) achieved similar results (collateral flow after RI was 21±2% of that in the normal zone). DPI or DETC blocked RI-induced p38 MAP kinase activation in response to VEGF and to RI, demonstrating a requirement for optimal ROS concentration in p38 MAP kinase activation. In addition, p38 MAP kinase inhibition prevented HCAEC tube formation and partially blocked RI-induced coronary collateral development (42±7% of normal zone flow, n=8) indicating that p38 MAP kinase is a critical signaling mediator of coronary collateral growth.