In voltage-dependent potassium channels, the molecular determinants of ion selectivity are found in the P (pore) region, a stretch of 21 contiguous residues. Cysteine was introduced at each P region position in a Shaker potassium channel. Residues projecting side chains into the pore were identified by means of channel inhibition by a sulfhydryl-reactive potassium ion analog, silver ion. The pattern of silver ion reactivity contradicts a beta barrel architecture of potassium channel pores.