In ruminants, endometrial prostaglandin F_2α (PGF_2α) is responsible for luteolysis and prostaglandin E₂ (PGE₂) is thought to be involved in maternal recognition of pregnancy. In the present study, healthy uteri were collected from cows at the abattoir, and days of the estrous cycle were determined macroscopically. The uteri were classified into seven groups as Days 1–3, 4–6, 7–9, 10–12, 13–15, 16–18, and 19–21 of the estrous cycle. Endometrial scrapings were collected. The expression of cyclooxygenase (COX)-1 and COX-2 mRNAs and proteins and PGE synthase (PGES) mRNA was analyzed by Northern and Western blot. There was no expression of COX-1, either mRNA or protein, on any day of the estrous cycle. In contrast, COX-2 mRNA and protein were expressed at low and high levels on Days 1–12 and 13–21 of the estrous cycle, respectively. The level of expression of PGES was moderate, low, and high on Days 1–3, 4–12, and 13–21 of the estrous cycle, respectively. There were significant correlations between COX-2 mRNA and protein levels and between COX-2 and PGES mRNA levels. COX-1 mRNA and protein are not expressed on any day of the estrous cycle, whereas COX-2 mRNA and protein and PGES mRNA are differentially expressed and regulated in bovine endometrium during the estrous cycle. COX-2, rather than COX-1, is the primary isoenzyme involved in the endometrial production of prostaglandins, and the COX-2 and PGES pathway is responsible for the endometrial production of PGE₂ in the bovine endometrium during the estrous cycle.