Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.