Protein sieving is a fundamental barrier property by which endothelial cells restrict transvascular protein flux. Proteins are thereby retained in blood, preventing excessive filtration, hence edema formation. Since the molecular basis of sieving is unknown, we considered the role of the junctional protein, E-cadherin, which is expressed by lung microvascular endothelial cells (ECs) (Quadri, JBC, 278:13342–9). We viewed cell junctions by confocal microscopy of EC monolayers expressing wild type (WT) E-cadherin-GFP, or a mutant E-cadherin-GFP (MT) that fails to form homotypic binding interactions in the E-cadherin ectodomain. To quantify permeability at junctional microdomains, we determined the z-axis fluorescence of 70-kD dextran (FD-70). In WT monolayers, junctional E-cadherin was distributed in high- (HD) and low- (LD) density microdomains. At HD microdomains, which comprised 60±5 % of the junctional length, FD-70 permeation was markedly lower than that at LD domains (P<0.05). In MT monolayers, the distribution of HD and LD domains and overall monolayer integrity, as quantified by electrical resistance, were similar to WT. However, the inter-domain permeability difference was abrogated (P<0.05). We conclude that homotypic interactions in the E-cadherin ectodomain determine macromolecular sieving by EC junctions (HL36024).