It is generally accepted that endothelin-1 (ET-1) is involved in the regression of the corpus luteum (CL) in cows, but there are few in vivo data available on the local release of vasoconstrictors, including ET-1. Thus, we aimed to determine in detail the local secretion of ET-1, angiotensin II (Ang II) and prostaglandin F2alpha (PGF2alpha) within the CL during spontaneous luteolysis in the cow. To observe real-time dynamics of the releasing profile of CL-derived factors, a microdialysis system was surgically implanted in the CL on day 15 of the estrous cycle and continuously perfused with Ringer's solution. Local secretion of ET-1, Ang II and PGF2alpha increased immediately after the onset of luteolysis (the time point when progesterone release started to decrease within the CL) and was maintained at high levels. A positive relationship was observed in intra-luteal changes among ET-1, Ang II and PGF2alpha release. This is the first real-time and in vivo evidence that the secretion of ET-1 together with Ang II and PGF2alpha immediately increases within the CL after the onset of spontaneous luteolysis. Consequently, we suggest that the activation of a local positive feedback mechanism among ET-1, Ang II and PGF2alpha might play a functional role in the paracrine modulation of luteolytic cascade and, simultaneously, the elevated ET-1 with Ang II and PGF2alpha should induce a strong vasoconstriction, thereby reducing the blood supplying the CL during spontaneous luteolysis.