Studies under blockade of alpha-, beta1-, and beta2-adrenoreceptors revealed a good correlation between the responses of rat colon relaxation of depolarized tonus and of rat adipocyte lipolysis elicited by catecholamines or BRL-37344, a selective beta3-adrenoreceptor agonist, suggesting beta3-adrenoreceptor stimulation. In contrast, SM-11044, a nonselective beta-adrenoreceptor agonist, stimulated colon relaxation more efficiently than lipolysis; its effects were differently antagonized by cyanopindolol with pA2 values of 8.31 in colon and of 7.32 in adipocytes. Binding studies in rat colon smooth muscle membranes using [125I]iodocyanopindolol under blockade of adrenaline and serotonin receptors revealed the existence of a single class of sites (Kd = 11.0 nM, Bmax = 716.7 fmol/mg protein). The specific binding was saturable and reversible and was displaced by SM-11044 but not by BRL-37344, isoproterenol, noradrenaline, adrenaline, serotonin, nor dopamine. This binding site was photoaffinity labeled using [125I]iodocyanopindolol-diazirine. The labeling was prevented by SM-11044 but not by BRL-37344. The amino-terminal amino acid sequences of the high performance liquid chromatography-purified peptides generated by enzymatic and chemical cleavages of the affinity labeled 34-kDa protein confirmed that the novel iodocyanopindolol or SM-11044 binding protein of rat colon smooth muscle membranes is different from known adrenaline, serotonin, or dopamine receptors. Its functional role might include the relaxation of depolarized colon.