The vascular leakage of macromolecules seen in several models after application of leukotriene B4 (LTB4) is mediated by neutrophil granulocytes. We describe here an in vitro assay for this event. Human umbilical vein endothelial cells were grown on polycarbonate filters separating luminal and abluminal compartments of fluid. Both clearance rate of fluorescein isothiocyanate albumin and neutrophil migration through the endothelial monolayer were increased when LTB4 (10-100 nM) was added to the abluminal compartment. However, if LTB4 was instead added to the luminal compartments together with the neutrophils, no migration or change in clearance could be detected. These findings were confirmed in vivo in the cheek pouches of anesthetized hamsters, where extravascular application of LTB4 induced intravascular adhesion of neutrophils, accompanied by neutrophil-dependent vascular leakage. On the other hand, intravascular deposition of LTB4 with micropipettes induced adhesion of leukocytes but no leakage. In conclusion, the presence of neutrophils adhering to endothelium does not necessarily imply the development of neutrophil-mediated vascular leakage. Instead, the leakage appears connected to the process of neutrophil chemotaxis.