Mannitol, which is a cell-impermeable and nontoxic polyalcohol, has been shown to be a useful tool for reversible opening of the blood-brain barrier (BBB). Despite successful clinical trials, the molecular mechanism of the mannitol-induced changes in cerebral endothelial cells (CECs) are poorly understood. For our experiments, we used CECs in culture, which were treated with different, clinically relevant concentrations of mannitol. We found that mannitol induced a rapid, concentration-dependent, and reversible tyrosine phosphorylation of a broad range of proteins between 50 and 190 kDa. One of the targets of tyrosine phosphorylation turned out to be the adherens junction protein b-catenin. Phosphorylation of b-catenin on tyrosine residues caused its subcellular redistribution and its dissociation from cadherin and a-catenin as shown by coimmunoprecipitation studies. All these effects could be inhibited by the Src kinase inhibitor PP-1 but not by the Erk inhibitor U0126, the Rho kinase inhibitor Y27632, or the calcium channel blocker verapamil. Because b-catenin is a key component of the junctional complex, its Src-mediated phpsphorylation may play an important role in the mannitol induced reversible opening of the BBB. V