The participation of cyclooxygenase (COX) in embryo implantation and parturition has been studied extensively. However, the distinct role of the two COX isoforms in these processes still remains unclear. Using three characterized mouse lines where the Ptgs1 and Ptgs2 genes substitute for one another, this study focused on the reproductive significance of their distinct roles and potential biological substitution. In both non-gravid and gravid uteri, the knock-in COX-2 is expressed constitutively, whereas the knock-in COX-1 is slightly induced in early implantation. The delayed onset of parturition previously found in COX-1 null mice was corrected by COX-2 exchange in COX-2>COX-1 mice, with normal term pregnancy, gestation length and litter size. In contrast, loss of native COX-2 in COX-1>COX-2 mice resulted in severely impaired reproductive functions. Knock-in COX-1 failed to substitute for the loss of COX-2 in COX-1>COX-2 mice during implantation, indicating that COX-1 may be replaced by COX-2, but not vice versa. A panel of prostaglandins detected in uterus and ovary demonstrates that prostaglandin biosynthesis preferentially depends on native COX-1, but not COX-2. More interestingly, preferential compensations by the COX isoforms were sustained despite weak dependency on their role in prostaglandin biosynthesis in the uterus and ovary.