Potassium channel inhibitor peptides from scorpion venom, α-KTx, have greatly advanced our understanding of potassium channel structure and function, Because of their high affinity interaction with the outer pore, α-KTx's have aided, in identification of amino acids lining the pore and of proteins constituting functional channels. The α-KTx's display a large range of affinities for different potassium channels with differences in binding free energy exceeding ∼8 kcal/mol. These differences in affinities are the foundation of α-KTx specificity and have aided in revealing the physiological and patho-physiological roles of potassium channels. The α-KTx subfamilies 1–3, display gross differences in specificity for maxi-K vs. KV channels. However, many potassium channels are largely untouched by α-KTx's. Differences in toxin binding free energy provide a quantitative framework for defining specificity. As a practical criterion for specificity a minimum binding free energy difference of 2.72 kcal/mol is proposed. Binding free energy differences for wild-type and mutant toxins and channels can point to amino acids underlying specificity and to unique features of potassium channel outer pores. Known 3D structures of potassium channels in combination with CLUSTALW sequence alignment of over 60 potassium channels reveal significant variation in α-KTx binding domains. Structure-based homology models of potassium channels complexed with α-KTxs, in combination with measurements of toxin binding free energy, will further our understanding of the molecular basis of α-KTx specificity.