Normal human endometrium (classified by histology and date after last menstrual period) was cultured for 72h, and the output of prostaglandin F 2α and 6-oxo-prostaglandin Fla detected by radioimmunoassay. Hormones/stimuli were added to the culture during the second day of culture for 5h and 19h periods. 1. 1) The output of prostaglandin F 2α from cultured endometrium was significantly higher (p<0.05) at the beginning (d4–8) and end (d25–30) of the menstrual cycle, compared to mid-cycle (d13–24) endometrium. Significantly more prostaglandin F 2α was released from proliferative than from secretory phase endometrium (p<0.02). 2. 2) Prostaglandin F 2α release was rapidly stimulated by sodium arachidonate (20–300 μg/ml), and by calcium ionophore A23187 (5 μg/ml) at an extracellular calcium ion concentration of 1.8mM.The ionophore stimulation was greater in mid-cycle endometrium than in endometrium from the beginning or the end of the menstrual cycle. 3. 3) Estradiol-17β (10 ng/ml) gradually increased the output of prostaglandin F 2α from secretory phase endometrium, and this stimulation was observed in the post-incubation period after hormone had been removed from the incubation medium. 4. 4) Oxytocin (1 × 10 −5U/ml caused a more rapid stimulation of prostaglandin F 2α output from secretory phase tissue (p<0.05 during the first 5h incubation period with hormone). 5. 5) Oxytocin (1 × 10 −5 U/ml) and estradiol (long/ml) together significantly stimulated prostaglandin F2a production by proliferative as well as secretory phase endometria. 6. 6) A high dose of hydrocortisone (loo μg/ml) inhibited the output of prostaglandin F 2α from proliferative and secretory phase endometrium and also from ionophore-stimulated endometrium. However, this dose of hydrocortisone did not inhibit the synthesis of prostaglandin F2a from exogenous arachidonic acid, or the estradiol-induced increase in prostaglandin F 2α production. 7. 7) Co-culture of endometrium with myometrium did not modify the output of prostaglandin F 2α or of 6-oxo-prostaglandin Fla from cultured tissues. 8. 8) These experiments suggest that arachidonic acid supply to the cyclooxygenase enzyme may vary during the menstrual cycle: and indicate a gradual increase in prostaglandin synthesising capacity in response to estrogen, more rapid control via oxytocin, and an interaction between estrogen and oxytocin to modulate prostaglandin F2a synthesis in human endometrium.