Mitogen-activated protein kinases (MAPKs) have been implicated in the signal transduction of the endothelial response to growth factors and inflammatory stimuli. The objective of this study was to test the hypothesis that the p42/44 MAPK pathway plays a common role in mediating the microvascular hyperpermeability response to vascular endothelial growth factor (VEGF) and histamine. The apparent permeability coefficient of albumin was measured in isolated and perfused coronary venules. Application of VEGF induced a rapid increase in venular permeability, and the effect was blocked by PD98059 and UO126, selective inhibitors of the mitogen-activated protein kinase kinase MEK1/2, in a dose-dependent pattern. The same MEK1/2 inhibitors dose-dependently attenuated the increase in venular permeability caused by histamine. In addition, the increases in venular permeability caused by agents that are known to activate the nitric oxide pathway, including the calcium ionophore ionomycin, the nitric oxide donor S-nitroso-N-acetylpenicillamine, and the protein kinase G activator 8-bromo-cGMP, were significantly attenuated in venules pretreated with the MEK1/2 inhibitors. Furthermore, transfection of venules with active MEK1 increased baseline permeability. In contrast, transfection of active ERK1, a downstream target of MEK1/2, did not significantly alter the basal permeability of venules. Moreover, inhibition of ERK1/2 with a specific inhibiting peptide did not prevent the hyperpermeability response to VEGF or histamine. The results suggest that activation of MEK1/2 may play a central role in the signal transduction of microvascular hyperpermeability in response to growth factors and inflammatory mediators.