The possible significance of the coexisting beta 1-, beta 2-, and beta 3-adrenoceptors in brown adipose tissue for the thermogenic response was investigated. Oxygen consumption of isolated hamster brown fat cells was analyzed as a measure of thermogenesis. Thermogenesis could be evoked not only by the physiological agent norepinephrine but also by BRL-37344 and CGP-12177. No evidence for biphasic inhibition curves was found with either the selective beta 1-antagonist ICI-89406, the beta 2-antagonist ICI-118551, or the beta 1/beta 2-nonselective beta-antagonist propranolol against 1 microM norepinephrine; pI50 (the negative logarithm of the inhibitory constant for an antagonist, as estimated from the dose-response curve for an antagonist vs. a constant agonist concentration) values for ICI-89406 and ICI-118551 were very low (4-5), implying nonselective inhibition; the pI50 for propranolol was approximately 6 (as expected for the beta 3-receptor). Even with suboptimal norepinephrine, no biphasic inhibition was found. CGP-12177 at concentrations where it is primarily an antagonist to the beta 1-receptor did not influence the dose-response curve for either norepinephrine or BRL-37344. BRL-37344- or CGP-12177-induced thermogenesis was inhibited by the beta-antagonists in a manner similar to norepinephrine-induced thermogenesis. Schild plots for propranolol inhibition of norepinephrine-, isoprenaline-, BRL-37344- and CGP-12177-induced thermogenesis yielded similar pA2 (the negative logarithm of the inhibitory constant for an antagonist, as calculated from a series of agonist dose-response curves at different antagonist concentrations) (approximately 5.5), for interaction with either agonist, implying that the same receptor was stimulated by all agonists. Thus, despite the fact that different beta-receptor subtypes coexist in the tissue, we find no evidence for the participation of beta 1- or beta 2-receptors in the thermogenic response. Within the resolution of the experiments, the results therefore imply that it is predominantly or solely the beta 3-receptor that is coupled to thermogenesis, and it is via this beta-adrenergic receptor that not only norepinephrine but also CGP-12177 and BRL-37344 induce thermogenesis.