The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A2 (iPLA2β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA2β-knockout (iPLA2β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I2 (PGI2), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI2 release and increased PAF production. Selective inhibition of iPLA2β with ( S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA2β-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI2 release. Stimulation of cardiac endothelial cells from group VIB (iPLA2γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI2 release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA2β activation and that both iPLA2β and iPLA2γ may be involved in PGI2 release.