Functional mouse prostaglandin E2 (PGE2) receptor EP3alpha subtype has been expressed in insect cells using the baculovirus system (C. Huang and H. H. Tai, 1995,Biochem . J. 307, 493-498). Site directed mutagenesis was carried out at Thr-221, Ser-268, and Ser-272. These hydroxy amino acid residues are highly conserved among prostaglandin receptors of many species and could potentially form hydrogen bonds with the hydroxyl group or the keto group of the receptor ligand PGE2. The mutant EP3alpha receptors again were expressed in insect cells and were studied by [3h]PGE2 binding assay. The results indicated that (1) replacement of Ser-272 by alanine did not cause any significant change in PGE2-binding activity, (2) replacement of Thr-221 by alanine also did not cause any significant change in PGE2-binding activity, and (3) replacement of Ser-268 by alanine almost abolished the PGE2-binding activity, while replacement by a threonine did not cause significant change in the PGE2-binding activity but did alter subtype specificity. These results suggest that Ser-268 of EP3alpha receptor plays an important role in ligand binding.