The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.