The Kir2.1 channel is characterized by strong inward rectification; however, the mechanism of the steep voltage dependence near the equilibrium potential remains to be investigated. Here, we studied the internal Ba(2+) block of the Kir2.1 channel expressed in Xenopus oocytes. We showed that the driving force and thus the K(+) ion flux significantly influenced the apparent affinity of the block by internal Ba(2+). Kinetic analysis revealed that the binding rate shifted with the driving force and changed steeply near the equilibrium point, either in the presence or absence of the transmembrane electrical field. The unbinding rate was determined by the intrinsic affinity of the site. Mutagenesis studies revealed that the high-affinity binding site for Ba(2+) was located near T141 at the internal entrance of the selectivity filter. The steep change of the blocking affinity near the equilibrium potential may result from the flux-coupling effect in the single-file, multi-ion cytoplasmic pore.