As no suitable radioligand exists for the detection of β₃-adrenoceptors, we have explored the radioligand binding properties of a tritiated version of the selective β₃-adrenoceptor antagonist L 748,337. Kinetic and equilibrium saturation and competition binding experiments were performed with [³H]-L 748,337 on membrane fractions of HEK and CHO cells stably transfected with human and rat β-adrenoceptor subtypes. Based on both association/dissociation kinetic and equilibrium saturation binding studies in transfected HEK cells, [³H]-L 748,337 exhibited an affinity of approximately 2 nM for human β₃-adrenoceptors. Competition studies with agonists and subtype-selective antagonists validated its binding to β₃-adrenoceptors. In CHO cells transfected with human β₃-adrenoceptors similar saturable high-affinity of [³H]-L 748,337 was observed. While some isoprenaline-sensitive [³H]-L 748,337 binding was also observed in CHO cells transfected with human β₁- or β₂-adrenoceptors, this was not saturable in a similar concentration range and/or not sensitive to the antagonists propranolol and SR 59,230, indicating that it did not primarily involve β-adrenoceptors. In CHO cells transfected with rat β₃-adrenoceptors [³H]-L 748,337 exhibited a considerably lower affinity than with the human subtype (12–95 nM). Low affinity for the rat β₃-adrenoceptor was also found with unlabelled L 748,337 in rat bladder strip relaxation experiments. We conclude that L 748,337 apparently has lower affinity for the rat than the human β₃-adrenoceptors and that [³H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of β-adrenoceptors. Nevertheless, [³H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human β₃-adrenoceptors reported to date.