Ba 2+ , a doubly charged analogue of K + , specifically blocks K + channels by virtue of electrostatic stabilization in the permeation pathway. Ba 2+ block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K + , the block-time distribution is double exponential, marking at least two Ba 2+ sites in the selectivity filter, in accord with a Ba 2+ -containing crystal structure of KcsA. By analyzing block as a function of extracellular K + , we determined the equilibrium dissociation constant of K + and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb + (3 µM) > Cs + (23 µM) > K + (29 µM) > NH 4 + (440 µM) >> Na + and Li + (>1 M). This represents an unusually high selectivity for K + over Na + , with |ΔΔG 0 | of at least 7 kcal mol −1 . These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.