The initiation of blastocyst implantation in the rat is indicated by localized increases in endometrial vascular permeability at the sites where blastocysts are present. The concentrations of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), a stable metabolite of prostaglandin I2 (PGI2), were measured by gas chromatography-mass spectrometry in the areas of increased endometrial vascular permeability (uterine dye sites), and compared with those in the remainder of the uterus (uterine non-dye sites). For rats killed either on the evening of Day 5 of pregnancy or on the morning of Day 6, measurable amounts of 6-keto-PGF1alpha were found in the dye sites of all animals, whereas 1 of 6 and 4 of 6 rats killed on Days 5 and 6, respectively, had undetectable amounts (less than 1 ng) in non-dye site tissue. It was estimated that, on average, the concentration of 6-keto-PGF1alpha in dye sites on the evening of Day 5 is at least 40-fold that in non-dye sites. The possible role of PGI2 in the initiation of blastocyst implantation is discussed.