In cattle, luteolysis results from the pulsatile release of endometrial prostaglandin F2α (PGF2α). Estradiol-17β (E2) and progesterone (P4) are involved in the regulation of luteolysis, but their mechanisms of action are unclear. The overall objective of this experiment was to investigate the actions of E2 and P4 on the control of PGF2α synthesis. Crossbred beef cows were slaughtered on Day 17 of a synchronized estrous cycle. Endometrial explants were incubated for 1 h with culture medium alone and for additional 11 h in culture medium supplemented with either 0, 10-12, or 10-11 M E2 (Experiment 1); 0, 10-9, 10-8, 10-7, 10-6 , or 10-5 M P4 (Experiment 2); or combinations of these concentrations (Experiment 3). Concentrations of PGF2α in culture medium were measured by a specific radioimmunoassay. Neither E2 (Experiment 1) nor P4 (Experiment 2) alone affected synthesis of PGF2α (P > 0.1). The combination of both steroids stimulated production of PGF2α in a concentration-dependent manner (P < 0.01). The greatest production of PGF2α was achieved when 10-11 M E2 was combined with 10-8 or 10-7 M P4. In conclusion, in vitro release of PGF2α was regulated by E2 and P4