We previously reported that Asn312 of the beta 2-adrenergic receptor and Asn385 in the homologous position in the 5-hydroxytryptamine1A receptor are important for binding to a class of beta-adrenergic receptor antagonists including propranolol and alprenolol. We proposed that the asparagine may be forming a hydrogen bond with the phenoxy oxygen common to these ligands. To further test this hypothesis we made alanine, threonine, phenylalanine, and glutamine substitutions at position 312 in the beta 2-adrenergic receptor. We observed that substitution with amino acids that permit formation of hydrogen bonds (threonine and glutamine) supported binding to aryloxyalkylamines, whereas substitution with amino acids that cannot form hydrogen bonds (alanine and phenylalanine) did not permit binding to these compounds. We were surprised to find that two of these substitutions led to an increase in affinity for alpha-adrenergic ligands. Substitution with glutamine and threonine at position 312 led to a 11-15-fold increase in affinity for yohimbine and enabled p-aminoclonidine to act as an agonist. These results further emphasize the role of position 312 in the formation of the ligand binding site for multiple ligands.