This article reports on the interaction of conducting (K(+)) and blocking (Na(+)) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K(+) channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA.K(+) and KcsA.Na(+) complexes of approximately 8 and 190 mm, respectively) and induce different conformations of the ion-bound protein. The differences in binding affinity as well as the higher K(+) concentration bathing the intracellular mouth of the channel, through which the cations gain access to the protein binding sites, should favor that only KcsA.K(+) complexes are formed under physiological-like conditions. Nevertheless, despite such prediction, it was also found that concentrations of Na(+) well below its dissociation constant and even in the presence of higher K(+) concentrations, cause a remarkable decrease in the protein thermal stability and facilitate thermal dissociation into subunits of the tetrameric KcsA, as concluded from the temperature dependence of the protein infrared spectra and from gel electrophoresis, respectively. These latter observations cannot be explained based on the occupancy of the binding sites from above and suggest that there must be additional ion binding sites, whose occupancy could not be detected by fluorescence and in which the affinity for Na(+) must be higher or at least similar to that of K(+). Moreover, cation binding as reported by means of fluorescence does not suffice to explain the large differences in free energy of stabilization involved in the formation of the KcsA.Na(+) and KcsA.K(+) complexes, which for the most part should arise from synergistic effects of the ion-mediated intersubunit interactions.