The present study investigates the action of zinterol at beta(3)-adrenoceptors. We used mouse primary brown adipocytes and Chinese hamster ovary (CHO-K1) cells expressing the mouse or human beta(3)-adrenoceptor. Zinterol was a full agonist at increasing cyclic AMP levels in primary brown adipocytes (which express beta(1)- and beta(3)-adrenoceptors but not beta(2)-adrenoceptors), and this effect was almost totally abolished in adipocytes derived from beta(3)-adrenoceptor knock-out (KO) mice. Zinterol was also a full agonist at increasing another biological end-point, glucose uptake in brown adipocytes. This effect was reduced in adipocytes derived from beta(3)-adrenoceptor KO mice, with the remaining response sensitive to beta(1)-adrenoceptor antagonism. To determine whether the effect of zinterol on beta(3)-adrenoceptors in primary brown adipocytes can be replicated in a recombinant system, we used CHO-K1 cells expressing the mouse or human beta(3)-adrenoceptor. Zinterol was a full agonist at mouse and human receptors with respect to increasing cyclic AMP levels, with pEC(50) values similar to that of the selective beta(3)-adrenoceptor agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate (CL316243) at the mouse receptor. At the human receptor, zinterol was more potent at increasing cyclic AMP levels than CL316243. In cytosensor microphysiometer studies, zinterol was a full agonist for increases in extracellular acidification rates at the mouse and human beta(3)-adrenoceptor. Therefore, we have shown that zinterol is a potent, high-efficacy beta(3)-adrenoceptor agonist at the endogenous mouse beta(3)-adrenoceptor in primary brown adipocytes and at the cloned mouse and human beta(3)-adrenoceptor expressed in CHO-K1 cells. Zinterol is therefore one of few beta-adrenoceptor agonists with high potency and efficacy at the human beta(3)-adrenoceptor.