Vascular Endothelial Growth Factor (VEGF) and histamine are well-known mediators of increased capillary permeability during various pathological conditions. We studied the acute effects of VEGF and histamine on the barrier function of Human Umbilical Vein Endothelial Cell (HUVEC) monolayers in vitro and the related intracellular signaling mechanisms. The resistance of HUVEC layer was measured by Electric Cell-substrate Impedance Sensing in which an AC current is applied and the resulting voltage drops across the cells (a few mV) is measured. Application of either histamine or VEGF gave a rapid, transient resistance decrease (lasting 2-3 min), which reflects a decrease of barrier function. The amplitude of the responses was concentration-dependent, VEGF: EC50 0.032 ± 0.001nM and histamine: EC50 3.3 ± 1.3nM. VEGF and histamine did not show an additive effect at maximal concentration and successive additions of either VEGF/histamine or histamine/VEGF abolished the second response. Both compounds elicited a transient increase in intracellular calcium levels, which correlated closely and inversely to the changes in barrier function. The response by histamine was mediated via H1-receptors; by VEGF via VEGF2-receptors. Chelation of intracellular calcium and inhibition of PKC, NO, ERK1/2, Rho kinase and PI3K, MLCK (myosin light chain kinase), tyrosine kinases, p38-MAPK all significantly attenuated both the VEGF and histamine response. Thus two different classes of receptors, histamine (G protein-coupled receptor) and VEGF (tyrosine kinase receptor) are able to convey the same action. They share the intracellular signaling pathways and acutely and transiently increase endothelial permeability with a comparable time course.