The specific binding of 125I-(-)pindolol to membranes prepared from rat colon displayed high affinity (Kd = 75 +/- 3 pM) and relatively low capacity (Bmax = 48 +/- 3.8 fmol/mg protein). Furthermore, the binding was reversible and possessed properties expected of a beta-adrenoceptor. Thus, for example, the potency of agonists in displacing 125I-(-)pindolol was, in decreasing order, isoproterenol greater than epinephrine greater than norepinephrine, and the negative isomer of propranolol was two orders of magnitude more potent than the positive isomer. Displacement of specific binding by the beta 1-adrenoceptor antagonist pafenolol and the beta 2-adrenoceptor antagonist ICI 118.551 revealed that the colon preparation possessed a heterogenous beta-adrenoceptor population. Analysis of the inhibition curves using computer-assisted curve fitting suggested that the sites consisted of a small (14%-21%) beta 1-adrenoceptor population and a large (79%-86%) beta 2-adrenoceptor population. The coexistence of beta 1- and beta 2-adrenoceptors is discussed in relation to evidence of a functional linkage of both subtypes with colon motility.