alpha-Dendrotoxin, a 59-amino acid basic peptide from the venom of Dendroaspis angusticeps (green mamba snake), potently blocks some but not all voltage-dependent potassium channels. Here we have investigated the relative contribution of the individual alpha-subunits constituting functional Kv1.1 potassium channels to alpha-dentroxin binding. Three residues critical for alpha-dentrotoxin binding and located in the loop between domains S5 and S6 were mutated (A352P, E353S, and Y379H), and multimeric cDNAs were constructed encoding homo- and heterotetrameric channels composed of all possible ratios of wild-type and mutant alpha-subunits. Complete mutant channels were about 200-fold less sensitive for the alpha-dendrotoxin block than complete wild-type channels, which is attributable to a smaller association rate. Analysis of the bimolecular reaction between alpha-dendrotoxin and the different homo- and heteromeric channel constructs revealed that the association rate depends on the number of wild-type alpha-subunits in the functional channel. Furthermore, we observed a linear relationship between the number of wild-type alpha-subunits in functional channels and the free energy for alpha-dendrotoxin binding, providing evidence that all four alpha-subunits must interact with alpha-dendrotoxin to produce a high affinity binding site.