1. We have studied the difference in receptor binding activity between partial and full beta2-adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the beta2-adrenoceptor, amino acid residues that are important for activation of the beta2-adrenoceptor. 2. In the binding study with [125I]-iodocyanopindolol, the Ki values of (+/-)-salbutamol, (+/-)-salmeterol, TA-2005 and (-)-isoprenaline for the beta2-adrenoceptor expressed in COS-7 cell membranes were 3340, 21.0, 12.0 and 904 nM, respectively. The beta1/beta2 selectivity of these agonists was in the order of (+/-)-salmeterol (332 fold) > TA-2005 (52.8) > (+/-)-salbutamol (6.8) > (-)-isoprenaline (1.1), and the beta3-/beta2-adrenoceptor selectivity of these agonists was in the order of TA-2005 (150 fold) > (+/-)-salmeterol (88.6) > (+/-)-salbutamol (10.4) > (-)-isoprenaline (3.2). 3. The maximal activation of adenylyl cyclase by stimulation of the beta1-, beta2- and beta3-adrenoceptors by TA-2005 was 32, 100 and 100% of that by (-)-isoprenaline, respectively, indicating that TA-2005 is a full agonist at the beta2- and beta3-adrenoceptors and a partial agonist at the beta1-adrenoceptor. (+/-)-Salbutamol and (+/-)-salmeterol were partial agonists at both beta1- (8% and 9% of (-)-isoprenaline) and beta2- (83% and 74% of (-)-isoprenaline) adrenoceptors. 4. The affinities of full agonists, TA-2005 and (-)-isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the beta2-adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (+/-)-salbutamol and (+/-)-salmeterol. Although the affinities of full agonists for the S207A-beta2-adrenoceptor were decreased, those of partial agonists for the S207A-beta2-adrenoceptor were essentially the same as for the wild type receptor. 5. The constitutively active mutant (L266S, L272A) of the beta2-adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the S207A-beta2-adrenoceptor were essentially the same as those for the wild type beta2-adrenoceptor, the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the constitutively active beta2-adrenoceptor were decreased by substitution of Ser207. 6. These results suggest that Ser204 and Ser207 of the wild type and constitutively active beta2-adrenoceptors differentially interacted with beta2-selective agonists.