Our previous study demonstrated that reactive oxygen species (ROS) released from activated blood cells contribute significantly to the increased microvessel permeability during inflammation. This study aims to define the individual roles of hydrogen peroxide, H₂O₂, and superoxide in ROS-induced increases in permeability and endothelial [Ca²⁺]_i in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca²⁺]_i was measured in fura-2-AM-loaded microvessels. Perfusing microvessels with superoxide generated by hypoxanthine and xanthine oxidase, HX/XO, induced immediate and transient increases in microvessel Lp. The mean peak value, occurred within 5 min of HX/XO exposure, was 4.3 ± 0.6 times that of the control. In contrast, perfusion of H₂O₂ (100 and 500 µM) caused no immediate increases in Lp. A significant Lp increase, 3.6 ± 0.6 times the control value, occurred 30 min after perfusion of H₂O₂ at 500 µM. The perfusion of H₂O₂ at 100 or 500 µM for 1h increased Lp to 6.6 ± 0.9 and 11.3 ± 3.6 times the control value, respectively. The increased endothelial [Ca²⁺]_i in HX/XO or H₂O₂ perfused vessels was correlated with the time course of the increases in Lp. Inhibiting Ca²⁺ influx by LaCl₃ prevented the permeability increase induced by HX/XO or H₂O₂. These results demonstrated differential actions of superoxide and H₂O₂ on microvessel permeability and endothelial [Ca²⁺]_i. Superoxide induced permeability increases were immediate and transient whereas H₂O₂ induced permeability increases were progressive, demonstrating concentration and time dependence. Calcium influx plays an essential role in both superoxide and H₂O₂-induced permeability increases.