This study tested the hypothesis that blood-borne prostaglandin F2α (PGF2α) produced at the time of ovulation by female goldfish, a typical scramble-spawning, egg-laying cyprinid fish, functions as a hormone which stimulates female sexual receptivity, behavior, and pheromone release, thereby synchronizing female mating behavior with egg availability. We conducted 5 experiments. First, we tested whether PGF2α is found in the blood of female fish and if it increases at the time of ovulation. Using gas chromatography–mass spectrometry, we found that circulating PGF2α was approximately 1 ng/ml prior to ovulation, increased over 50-fold within 3 h of ovulation and returned to preovulatory values after spawning and egg release. Ovulated fish also released over 2 ng/h of PGF2α and 800 ng/h of 15-keto-PGF2α, a metabolite of PGF2α – both compounds with known pheromonal function. Second, we tested how closely levels of circulating PGF2α tracked the timing of ovulation by sampling fish at the time of ovulation and discovered that PGF2α increased within 15 min of ovulation, peaked after 9 h, and fell to basal levels as fish spawned and released their eggs. Third, we tested whether an interaction between eggs and the reproductive tract serves as a source of circulating PGF2α and its relationship with female sexual receptivity by injecting ovulated eggs (or an egg-substitute) into the reproductive tract of females stripped of ovulated eggs. We found both of these treatments elicited measurable increases in plasma PGF2α as well as female sexual behavior. A fourth experiment showed that indothemacin, a PG synthase inhibitor, blocked both PGF2α increase and female sexual behavior in egg-substitute-injected fish. Finally, we tested the relationship between the expression of female behavior and PGF2α in PGF2α-injected fish and found that circulating PGF2α levels closely paralleled behavior, rising within 15 min and peaking at 45 min. Together, these experiments establish that PGF2α functions as a behavioral blood-borne hormone in the goldfish, suggesting it likely has similar activity in other related, externally-fertilizing fishes.