KAT1 is a cloned voltage-gated K+ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of 'anomalous' rectification of the if pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb+ solution than in pure K+ solution, and negligible in pure Na+ solution. Experiments in different K+/Na+ or K+/Rb+ mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K+/Rb+ mole fraction (i.e., the ratio [K+]/([K+]+[Rb+])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called 'foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K+/Rb+ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in PRb/PK. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical 'green' member of the Shaker superfamily of K+ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies.