Abstract Differentiation of a restrictive endothelial barrier in the chick chorioallantoic membrane (CAM) occurs between Day 4.5 and Day 5.0 of the normal 21-day gestation. Whether molecular changes in the endothelial cell-cell junctional protein complex contribute to the ontogeny of barrier function represents the principal focus of this study. VE-cadherin has been shown to contribute to the regulation of endothelial cell monolayer permeability in vitro. Accordingly, VE-cadherin is complexed to the cytosolic catenins, and changes in monolayer permeability have been linked to alterations of the cadherin/catenin complex. Currently, a CAM endothelial VE-cadherin/β-catenin complex was identified, and phosphotyrosine labeling of β-catenin was decreased concurrently with the abrupt increase in CAM endothelial selectivity between Day 4.5 and Day 5.0. Further, inhibition of protein tyrosine phosphatases impeded regular tyrosine dephosphorylation of β-catenin at Day 5.0 and this served to partially restore macromolecular extravasation to elevated levels normally present at Day 4.5. Thus, differentiation of selective barrier function in the angiogenic CAM endothelium in vivo is dependent, in part, on tyrosine dephosphorylation of β-catenin.