Accumulating evidence indicates that overproduction of prostanoids attributable to overexpression of cyclooxygenase-2 (COX-2) plays an important role in colon carcinogenesis. We have shown recently that the prostaglandin (PG) E receptor, EP₁, but not EP₃, is involved in mouse colon carcinogenesis. In line with our previous study, here we examined the role of prostanoid receptors in colon carcinogenesis using six additional lines of knockout mice deficient in prostanoid receptors EP₂, EP₄, DP, FP, IP, or TP. The animals were treated with the colon carcinogen, azoxymethane (AOM), and examined for the development of aberrant crypt foci (ACFs), putative preneoplastic lesions in the colon. Formation of ACFs was decreased only in the EP₄-knockout mice, to 56% of the wild-type level. To confirm these results, we also examined the inhibitory effects of an EP₄-selective antagonist, ONO-AE2-227, in the diet on the formation of AOM-induced colon ACFs in C57BL/6Cr mice and on the development of intestinal polyps in Min mice. ONO-AE2-227 at a dose of 400 ppm reduced the formation of ACFs to 67% of the control level, and intestinal polyp numbers in Min mice receiving 300 ppm were decreased to 69% of the control level. Plating efficiency assays showed that addition of 1.0 μm ONO-AE1-329, an EP₄-selective agonist, resulted in a 1.8-fold increase in the colony number of the human colon cancer cell line, HCA-7, similar to the effect of PGE₂. Moreover, EP₄ mRNA expression was clearly observed in normal colon mucosa and colon tumors in mice. Our previous and present results indicate that PGE₂ contributes to colon carcinogenesis through its actions mediated through EP₁ and EP₄ receptors; therefore, antagonists for these two receptors may be good candidates as chemopreventive agents against colon cancer.