Recent mutagenesis studies of the ion channel proteins have allowed us to identify amino acid residues critical in determining ion selectivity. Ion selectivity of a channel can be altered even by single amino acid substitutions. Functional analyses of mutants largely support views in classical biophysics that the pore size and the fixed charges are major determinants of ion selectivity. For full understanding of the molecular mechanism of ion selectivity, elucidation of the tertiary structure of channel proteins remains essential.