We studied the binding region of several beta1 and beta2 selective agonists by using chimeric beta1 and beta2ARs, and point-mutated beta2 adrenergic receptors (ARs). By replacing a single transmembrane domain (TMD) of beta1AR (or beta2AR) with the corresponding region of beta2AR (or beta1AR), we found that beta2 or beta1 selectivities were determined by TMD2 and TMD7 of beta2AR or by TMD2 of beta1AR, respectively. Alanine-substituted beta2AR mutants showed that tyrosine at position 308 in TMD7 played an important role in binding of beta2 selective agonists with high affinity. These data also suggested that the substituent on the amine portion was important for subtype selective agonist binding.