We investigated the permeability changes that occur in the human brain microvascular endothelial cell (HBMEC) monolayer, an in vitro model of the blood-brain barrier, during Escherichia coli K1 infection. An increase in permeability of HBMECs and a decrease in transendothelial electrical resistance were observed. These permeability changes occurred only when HBMECs were infected with E. coli expressing outer membrane protein A (OmpA) and preceded the traversal of bacteria across the monolayer. Activated protein kinase C (PKC)–α interacts with vascular-endothelial cadherins (VECs) at the tight junctions of HBMECs, resulting in the dissociation of β-catenins from VECs and leading to the increased permeability of the HBMEC monolayer. Overexpression of a dominant negative form of PKC-α in HBMECs blocked the E. coli–induced increase in permeability of HBMECs. Anti-OmpA and anti–OmpA receptor antibodies exerted inhibition of E. coli–induced permeability of HBMEC monolayers. This inhibition was the result of the absence of PKC-α activation in HBMECs treated with the antibodies