In this study, a mutagenesis-based strategy was employed to assess the roles of two highly conserved motifs (KLR and RLAR) within the third endoloop of the human secretin receptor. Block deletion of KLRT and mutation of Lys323 (K(323)I) significantly reduced cAMP accumulation, and these mutations did not affect ligand interaction and receptor number expressed on the cell surface. Thus, the KLRT region at the N terminus of the third endoloop, particularly Lys323, is important for G protein coupling. For the RLAR motif, receptors with substitutions at positions 339 and 342 from Arg to Ala (R(339, 342)A), Glu (R(339, 342)E), or Ile (R(339, 342)I) as well as block deletion of the RLAR motif were all found to be defective in both secretin-binding and cAMP production. Interestingly, a single mutation at the corresponding positions of Arg339 or Arg342 responded as the wild-type human secretin receptor in all functional assays, indicating that the presence of one Arg at either position within the RLAR motif is sufficient for a normal receptor function. Immunofluorescent staining of these mutant receptors showed that these Arg residues are responsible for surface presentation and/or receptor stability.