Kat1 is a highly selective inward-rectifying K(+) channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K(+) channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K(+) over Na(+). Mutants of this class frequently displayed sensitivity to the competing ion Cs(+). This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K(+)/Na(+) selectivity, but selectivity over Cs(+), and possibly a wide spectrum of potential competing ions.