Angiotensin II (Ang II), a potent modulator of cardiovascular homeostasis, mediates its effects through two Ang II receptor subtypes (AT1 and AT2) which share less than 30% amino acid homology and can be differentiated by specific nonpeptide ligands. We investigated the structural determinants of ligand binding to the AT1 receptor for L-163,017 which belongs to a newly developed class of nonpeptides that have equivalent affinities for AT1 and AT2 receptors. Nonpeptide binding affinities were determined in radioreceptor binding assays in membranes from COS cells transfected with wild-type and mutant receptor cDNAs. The amphibian AT receptor variant, xCM46, recognized L-163,017 with an affinity (IC50=8±2 nM) that was only 1.5-fold lower than for the rat AT1 receptor (IC50=5±1 nM) which is in striking contrast to the poor affinity of the amphibian xATa wild-type receptor (IC50 >50 µM). Analysis of single point rAT1 receptor mutants in which individual mammalian residues were replaced by the corresponding frog amino acids revealed significant overlap but also distinct differences in ligand binding interactions between dual receptor and subtype selective non-peptides. These data suggest that AT1-selective and dual receptor nonpeptides share overlapping but distinct binding pockets on the AT1 receptor. These findings may lead to improved therapeutics for the treatment of cardiovascular diseases involving both AT receptor subtypes.