Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration and angiogenesis. EETs play a role in the VEGF-activated signal transduction cascade but the aim of this study was to assess their effect on the stabilization of endothelial cell tubes. Stimulation with VEGF increased CYP2C9 promoter activity in endothelial cells and enhanced CYP2C mRNA and protein expression resulting in increased intracellular EET levels. Moreover, VEGF-induced endothelial cell sprouting was reduced by CYP2C antisense oligonucleotides as well as by the EET-antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE). The antagonist did not affect basic fibroblast growth factor-stimulated tube formation. In vivo (Matrigel plug assay, 10 days), VEGF elicited the formation of endothelial cell tubes that lacked significant smooth muscle cell/pericyte coating; an effect sensitive to 14,15-EEZE. However, vessels formed in EET-impregnated plugs were coated with smooth muscle cells/pericytes, and significantly perfused as indicated by contrast-enhanced ultrasound. As both platelet-derived growth factor (PDGF) b and the sphingosine 1 phosphate 1 (S1P1) receptor have been implicated in pericyte recruitment and endothelial cell coverage we determined possible interactions with 11,12-EET. Overexpression of CYP2C9 in human endothelial cells increased the expression of the S1P1 receptor and increased the S1P-induced endothelial cell hyperpolarization in a CYP inhibitor-dependent manner. 11,12-EET was also able to increase expression of the PDGF receptor in cultured endothelial cells as well as in the inner lining of the vessels formed in Matrigel plugs. Taken together, our data indicate that CYP2C-derived EETs not only participate as second messengers in the angiogenic response initiated by VEGF, but that EETs have the potential to influence much more than angiogenesis by enhancing pericyte recruitment to endothelial cell tubes to promote vascular maturation. The latter depends on the activation of S1P and PDGF receptors.