The outputs of PGF2 alpha, PGE2 and 6-oxo-PGF1 alpha from the uterus of ovariectomized guinea-pigs were all low (approximately 0.1 to 0.2 ng/min/uterine horn). PGF2 alpha was released in greatest quantities, followed by PGE2 and then by 6-oxo-PGF1 alpha. Progesterone treatment had no effect on PG output. Estradiol treatment increased PGF2 alpha output 7-fold, but increased PGE2 and 6-oxo-PGF1 alpha outputs only 1.7-fold and 2.9-fold, respectively. Estradiol treatment reversed the PGE2 to 6-oxo-PGF1 alpha ratio. Progesterone-priming before estradiol treatment potentiated the stimulatory effect of estradiol on PGF2 alpha release up to 2-fold, but had no such action on the stimulatory effect of estradiol on PGE2 and 6-oxo-PGF1 alpha release. Estradiol acting on a progesterone-primed uterus causes a relatively specific activation of PGF2 alpha synthesis and release. The physiological significance of this observation is discussed. A23187 stimulated PGF2 alpha release from all uteri irrespective of treatment. However, the magnitude of the response was much greater in those guinea-pigs which had received estradiol (either alone or with progesterone) than in those which had not. A23187 stimulated also 6-oxo-PGF1 alpha release, but to a much lesser extent, and had no effect on PGE2 release. Raising free intracellular Ca2+ levels in the uterus stimulates PGF2 alpha release and, to a lesser extent, 6-oxo-PGF1 alpha release, but the size of the response depends upon the hormonal status of the animal.