Prostaglandin F(2)(alpha) (PGF(2)(alpha)) released from the uterus causes alterations in luteal blood flow, reduces progesterone secretion, and induces luteolysis in the bovine corpus luteum (CL). We have recently discovered that luteal blood flow in the periphery of the mature CL acutely increases coincidently with pulsatile increases in a metabolite of PGF(2)(alpha) (PGFM). In this study, we characterized changes in regional luteal blood flow together with regional alterations in endothelial nitric oxide synthase (eNOS) expression during spontaneous luteolysis and in response to PGF(2)(alpha). Smooth muscle actin-positive blood vessels larger than 20 microm were observed mainly in the periphery of mature CL. PGF(2)(alpha) receptor was localized to luteal cells and large blood vessels in the periphery of mid-CL. PGF(2)(alpha) acutely stimulated eNOS expression in the periphery but not in the center of mature CL. Injection of the NO donor S-nitroso-N-acetylpenicillamine into CL induced an acute increase in luteal blood flow and shortened the estrous cycle. In contrast, injection of the NOS inhibitor l-NAME into CL completely suppressed the acute increase in luteal blood flow induced by PGF(2)(alpha) and delayed the onset of luteolysis. In conclusion, PGF(2)(alpha) has a site-restricted action depending on not only luteal phase but also the region in the CL. PGF(2)(alpha) stimulates eNOS expression, vasodilation of blood vessels, and increased luteal blood flow in periphery of mature CL. Furthermore, the increased blood flow is mediated by NO, suggesting that the acute increase in peripheral blood flow to CL is one of the first physiological indicators of NO action in response to PGF(2)(alpha).