The prostaglandin EP3 receptor binds Prostaglandin E2 in a ligand binding pocket formed in part by seven transmembrane alpha-helices. The present studies demonstrate that the second extracellular loop of the receptor is involved in prostanoid ligand recognition as well. Site-directed mutagenesis of seven conserved residues clustered in the amino portion of the second extracellular loop was performed. Receptors with single amino acid substitutions at each of these positions were transiently transfected into HEK293tsA201 cells, their ligand binding profiles assessed, and each receptor was tested for its ability to decrease intracellular cAMP levels. Substitution of Trp199 or Thr202 with alanine resulted in receptors with increases in affinity up to 128-fold for prostanoid compounds with a C1 methyl ester but wild type affinities for natural prostanoid ligands that have a carboxylate moiety at the C1 position. In contrast, substitution of Pro200 with serine caused a loss of selectivity up to 20-fold for naturally occurring prostanoid agonists as compared with the wild type EP3 receptor: the PS200 receptor displayed a decrease in affinity for E-ring compounds and an increase in affinity for F- and D-ring compounds. The EC50 for inhibition of cAMP remained unchanged for each receptor tested.