Abstract The β-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily β 1 (atria)-, β 2 (trachea)- and atypical β β 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (−)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (±)-Trimetoquinol was equally or slightly less active than (−)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of β-adrenoceptor subtypes. In functional assays, 3′-iodotrimetoquinol was a potent activator of all β-adrenoceptor subtypes. 3′,5′-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical β β 3 - adrenoceptors than those tissues containing β 1- and β 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (±)-trimetoquinol and 3′-iodotrimetoquinol on β 1-adrenoceptors. Pharmacological properties of the compounds on rat β 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3′,5′-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat β 3-adrenoceptor was 3′-iodotrimetoquinol = 3′,5′-diiodotrimetoquinol > (±)-trimetoquinol > (−)-isoprenaline. These results suggest that 3′,5′-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective β 3-adrenoceptor ligands.