Although tight-junctions (TJs) at the blood-brain barrier (BBB) are important to prevent non-specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR-BBB13), type II astrocyte (TR-AST4) and brain pericyte (TR-PCT1) cell lines. Transfilter co-culture with TR-AST4 cells, and exposure to conditioned medium of TR-AST4 cells (AST-CM) or TR-PCT1 cells (PCT-CM) increased occludin mRNA in TR-BBB13 cells. PCT-CM-induced occludin up-regulation was significantly inhibited by an angiopoietin-1-neutralizing antibody, whereas the up-regulation by AST-CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin-1 is secreted from TR-PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie-2 in TR-BBB13 cells. A fractionated AST-CM study revealed that factors in the molecular weight range of 30-100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor beta 1, the mRNA of which was up-regulated in TR-AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte-derived multimeric angiopoietin-1/Tie-2 pathway induces occludin expression.