The enzymatic products of cyclo-oxygenase (COX) activity regulate angiogenesis in vitro and in vivo, implicating altered COX expression and prostaglandin (PG) synthesis in the actions of pro-angiogenic mediators. We have previously shown that inhibition of induced COX-2 activity decreases endothelial cell (EC) tube formation in vitro and that this is rescued by exogenous iloprost (PGI2 analogue), suggesting an autocrine mode of action. PGI2 binds to the cell surface GPCR, IP, and to the ligand-activated nuclear receptor peroxisome proliferator activated receptor (PPAR) β/δ. Here, we have determined the relative contributions of PPARβ/δ and IP to EC function and angiogenesis. Iloprost, cicaprost (binds IP) and selective PPARβ/δ agonists (GW501516; L-165,041) increased neovascularisation in vivo (chick CAM assay; p<0.05 iloprost and cicaprost n=10-13, and p<0.001 PPARβ/δ n=17-26) and EC tubulogenesis on Matrigel in vitro (p<0.05 for all agonists; n=3). The PPARβ/δ antagonist GSK0660 abrogated iloprost- and PPARβ/δ agonist-induced tube formation. The angiogenic responses to thrombin and to protease-activated receptor agonist peptides (PAR2-AP and PAR4-AP) were attenuated by GSK0660 (p<0.01 for all agonists; n=3) and by PPARβ/δ siRNA but not by a specific IP antagonist, CAY10441. Immunoblotting studies showed that thrombin and PAR2-AP, but not PAR4-AP, enhance COX-2 expression and that this is reduced by antagonists of PPARβ/δ but not IP (n=3). EC exposure to thrombin or to PPARβ/δ agonists promoted acute activation of ERK, AMPK and Akt, as well as induction of COX-2 expression, all of which were inhibited by PPARβ/δ blockade (n=3). GSK0660 also reduced phosphorylation of Akt, AMPK and ERK in response to thrombin (n=3). These data implicate PPARβ/δ in the regulation of COX-2 induction by thrombin and PAR2-AP, and in the consequent tubulogenesis, and suggest that PPARβ/δ propagates the angiogenic response through an autocrine feedback loop. The observation that GSK0660 suppressed PAR4-AP-driven tube formation indicates that PPARβ/δ also regulates tubulogenesis through COX-2-independent mechanisms. Together, these studies provide evidence of a key role for PPARβ/δ in mediating agonist-induced angiogenesis.