Extracellular nucleotides play important roles in inflammation. We previously found that nucleotide P2Y2 receptor activation induced transient 5-fold increase in venular permeability to albumin (Ps) from baseline in wild type (WT) mice, but not in P2Y2 R knockout (P2Y2 R KO) mice. The objective of this study is to investigate the role of focal adhesion kinase (FAK) in P2Y2 R signaling-dependent modulation of endothelial permeability. Primary cultured microvascular endothelial cells (MEC) of skeletal muscles, derived from either WT or P2Y2 R KO mice, were used in this study. Immunoblot analysis revealed increase in FAK activity of 1.48±0.11 (n=3) and 1.58±0.08 (n=3) at 10 min and 30 min UTP (an agonist of P2Y2R; 10-5 M), respectively, in WT MEC. In contrast, FAK activity didn't change in P2Y2R KO MEC. Activity of VE-cadherin, a key molecule of the adherens junction, was assessed via measuring tyrosine phosphorylation of VE-cadherin at tyrosine 658 (pY658) in HUVEC. UTP (10-5 M) induced 1.16±0.03 (n=4) and 1.19±0.07 (n=4) increase in pY658 level of VE-cadherin at 5 and 15 min, respectively, compared to the control group. The findings support the hypothesis that FAK activation is associated with P2Y2 R-induced increased microvascular permeability. (Supported by Missouri State University Research Funding).