AbstractObjectiveThe mechanisms by which histamine increases microvascular permeability remain poorly understood. We tested the hypothesis that H1 receptor activation disrupts the endothelial barrier and investigated potential downstream signals.MethodsWe used confluentECmonolayers, assessingTERas an index of barrier function.HUVEC,HCMEC, andHDMECwere compared. Receptor expression was investigated using Western blotting,IFconfocal microscopy andRT‐PCR. Receptor function and downstream signaling pathways were tested using pharmacologic antagonists and inhibitors, respectively.ResultsWe identified H1–H4 receptors on all threeECtypes. H1 antagonists did not affect basalTERbut prevented the histamine‐induced decrease inTER. Blockade of H2 or H3 attenuated the histamine response only inHDMEC, while inhibition of H4 attenuated the response only inHUVEC. Combined inhibition of bothPKCand PI3K caused exaggerated histamine‐induced barrier dysfunction inHDMEC, whereas inhibition of p38MAPkinase attenuated the histamine response in all threeECtypes. Inhibition of RhoA,ROCK, orMLCKalso prevented the histamine‐induced decrease inTERinHDMEC.ConclusionThe data suggest that multiple signaling pathways contribute to histamine‐induced endothelial barrier dysfunction via the H1 receptor.