Three isoforms of the mouse prostaglandin‐E‐receptor EP₃ subtype (EP₃), EP_3α, EP_3β, and EP_3γ, with different C‐termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP‐binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S. & Ichikawa, A. (1993) J. Biol. Chem. 268, 2712–2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S. & Ichikawa, A. (1993) Eur. J. Biochem. 217, 313–318]. To assess the role of the C‐terminus in GTP‐binding protein coupling, we truncated the C‐terminus of EP₃ at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with G_i2, forming an agonist/receptor/G_i2 ternary complex, and to undergo the characteristic conversion of its agonist‐binding affinity, mediated by a guanine nucleotide from a low‐affinity state to a high‐affinity state. However, sulprostone, an EP₃ agonist, failed not only to inhibit the forskolin‐induced CAMP accumulation in the mutant receptor‐expressing cells but also to stimulate the GTPase activity in the mutant receptor‐expressing cell membrane. These results indicated that the C‐terminus of EP₃ is essential for the activation of GTP‐binding protein.