alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha 1a-AR, alpha 1b-AR, alpha 1d-AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha 1b- or alpha 1d- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha 1b-AR were converted, either individually or in combination, to the corresponding residues in the alpha 1a-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha 1a-AR. Over 80% of this conversion was due to an Ala204-->Val substitution; the remainder was due to the additional substitution of Leu314-->Met. To confirm that Ala204 and Leu314 are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha 1a-AR (Val185 and Met293) were reversed of that of the alpha 1b-AR. Correspondingly, the agonist-binding profile of this double alpha 1a-AR mutant reverted to that of the alpha 1b-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.