This study aimed to determine whether prostaglandin D2 (PGD2) is a major uterine cyclooxygenase (COX) product and if so, we wanted to examine the underlying mechanism, its relation to COX-1-mediated metabolism, and how it influences the in vitro myometrial contraction during the late stage of pregnancy. The production of PGD2 or responses evoked by the prostanoid were determined in uteri isolated from prepartum and/or non-pregnant C57Bl/6 wild-type (WT) or COX-1-/- mice. Results showed that PGD2, which was not detected in non-pregnant counterparts, appears as the major prostanoid in prepartum (<24 h prior to parturition) mouse uteri. No signal of PGD2 or other COX-derived products was detected in similar tissues of COX-1−/− mice. Western blot or real-time PCR revealed that expressions of COX-1 and PGD2 synthase (PGDS) in prepartum uteri were higher than those of non-pregnant mice, while both were diminished by the removal of endometrium. Also, we noted that in endometrium-removed prepartum uteri PGD2 evoked an increased contraction compared to that of non-pregnant mice. Antagonizing the F prostanoid (FP) receptor but not D prostanoid receptors abolished the contraction. Moreover, the level of FP receptor mRNAs in endometrium-removed prepartum uteri was increased compared to that of non-pregnant mice. These results imply that due to up-regulations of COX-1 and PGDS in endometrium, PGD2 becomes the major prostanoid produced in prepartum uteri where it can evoke an increased in vitro myometrial contraction, possibly resulting from up-regulation of the FP receptor, the mediator of such a response in mouse uteri.