To explore the role of prostaglandin E(2) (PGE(2)) in angiogenesis in the developing corpus luteum, luteal microvascular endothelial-like cells (luteal ECs) were prepared from highly luteinizing ovaries of rats using the percoll density gradient method. The cells abundantly expressed the mRNAs of the endothelial markers CD31 (PECAM-1) and responded to the vascular endothelial growth factor (VEGF) to form in vitro tube structures on Matrigel. Cyclooxygenase (COX) inhibitors significantly suppressed tube formation in luteal ECs, whereas PGE(2) counteracted the COX inhibitor-induced blockage. PGE(2)-induced tube formation was blocked by a cyclic AMP-dependent protein kinase A (PKA) inhibitor, H89. The antagonist against the PGE receptor type 2 (EP2 receptor), AH6809, completely inhibited PGE(2)-induced tube formation and partly suppressed the VEGF-induced tube formation but did not attenuate PGE(2)-induced phosphorylation of both AKT kinase and extracellular signal-regulated kinase 1/2. VEGF significantly enhanced the expression of COX-2 mRNAs detected by real-time RT-PCR and PGE(2) secretion into the media measured by ELISA in luteal ECs. In turn, PGE(2) stimulated VEGF expression. In vitro co-culture of luteal ECs with steroidogenic luteal cells (SLCs) promoted tube formation. Pre-treatment of SLCs with VEGF further enhanced tube formation of ECs, and this effect was blocked by the COX-2 inhibitor. This stimulatory effect was inhibited by treatment with AH6809. These data indicate that PGE(2) exerts a direct stimulatory effect on tube formation mainly via the EP2 receptor/PKA signaling in luteal ECs. Our results suggest the possibility that the endogenous PGE(2) that is produced from luteinizing follicular cells as well as ECs may stimulate luteal angiogenesis.