The effects of 17 beta-estradiol on the levels of platelet-activating factor (PAF) and prostaglandins and their precursor phospholipid in the uterus of oophorectomized rats were studied. Oophorectomy results in the decrease in the uterine PAF level to one-third of that in natural estrus. This level was recovered by subcutaneous administration of 17 beta-estradiol. The level of uterine phospholipids, which are rich in arachidonic acid, was significantly decreased by estradiol treatment. More arachidonate-PC was depleted than arachidonate-PE. The molecular structure was confirmed by gas chromatography-mass spectrometry. The amount of PGF2 alpha in the oophorectomized uterine tissue was 10-times that of PAF, but like the latter, increased 3-4 times on estradiol treatment. The chemical structures of PAF and PGF2 alpha formed on estradiol treatment were confirmed by mass spectrometry. The present data strongly suggest a correlation between the formations of PAF and PGF2 alpha, and indicate that estradiol may regulate the physiological formations of PAF and PGs in non-pregnant rat uterus.