The contribution of the extracellular loops of the secretin receptor to the recognition of secretin was investigated by transfection in CHO cells of chimeric receptors, in which the three loops of the secretin recombinant receptor were replaced by the corresponding sequences of the glucagon receptor. The role of the third loop could not be evaluated as the transfected cells did not respond to secretin. Replacement of extracellular loop 2 reduced markedly the capability of secretin to occupy the receptor but did not alter the capacity of the receptor to discriminate between peptide analogues modified in position 3. Replacement of the first extracellular loop not only reduced the potency of secretin but also decreased the capacity of the receptor to discriminate between ligands having in position 3 an aspartate (as in secretin), an asparagine, or a glutamic acid. This change in receptor properties was reproduced by a single mutation of lysine 173 of the receptor into isoleucine. Thus, the basic amino acid in position 173 is likely to interact with aspartate 3 of secretin. As an aspartate is also present in position 3 of VIP and PACAP, two peptides related to secretin, and a lysine residue is conserved in the first extracellular loop of the VIP and PACAP receptors, this interaction may be a key element of peptide recognition by this receptor family.