Our previous study demonstrated that TNF- induced significant leukocyte adhesion without causing increases in microvessel permeability, and that fMLP-stimulated neutrophils in the absence of adhesion increased microvessel permeability via released reactive oxygen species (ROS). The objective of our present study is to investigate the mechanisms that regulate neutrophil respiratory burst and the roles of fMLP-stimulated ROS release from adherent leukocytes in microvessel permeability. A technique that combines single microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-. Microvessel permeability was assessed by measuring hydraulic conductivity (Lp). The autologous blood perfusion after TNF- application for 2h increased leukocyte adhesion from 1.2 ± 0.2 to 13.3 ± 1.6/100 µm of vessel length without causing increases in Lp. When fMLP (10 µM) was applied to either perfusate (n=5) or superfusate (n=8) in the presence of adherent leukocytes, Lp transiently increased to 4.9 ± 0.9 and 4.4 ± 0.3 times the control value, respectively. Application of superoxide dismutase, or an iron chelator, deferoxamine mesylate, prior to fMLP application prevented or attenuated the Lp increase. Chemiluminescence measurements in isolated neutrophils demonstrated that TNF- alone did not induce ROS release but pre-exposure of neutrophils to TNF- in vivo or in vitro potentiated fMLP-stimulated ROS release. These results suggest a priming role of TNF- in fMLP-stimulated neutrophil respiratory burst, and indicate that the released ROS play a key role in leukocyte-mediated permeability increases during acute inflammation.