The differential role of amino acids of the third transmembrane domain on peptide and nonpeptide recognition by the AT1 angiotensin II receptor has been evidenced. The mutation of Ser105 into alanine completely abolished peptide agonist and antagonist binding, while the binding of nonpeptide ligands, including the original radioligands [3H] LF 7-0156 and [3H] LF 8-0129, was more moderately affected. Reverse pharmacological changes, i.e., unchanged affinities for peptide agonists or antagonists and drastically reduced affinities for nonpeptide antagonists, were observed upon alanine replacement of Asn111. These results confirm that the binding sites for peptide and nonpeptide molecules are not totally overlapping and delineate new amino acids as candidates for the selective receptor interaction with the two categories of ligands. Their integration in topographical studies is discussed.