The Arabidopsis thaliana cDNA, KAT1, encodes a hyperpolarization- activated K+ channel. In the present study, we utilized a combination of random site-directed mutagenesis, genetic screening in a potassium uptake- deficient yeast strain, and electrophysiological analysis in Xenopus oocytes to identify strong modifications in cation selectivity of the inward rectifying K+ channel KAT1. Threonine at position 256 was replaced by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K+ uptake-deficient yeast strain at low concentrations of potassium. Among these, two mutants (T256D and T256G) showed a sensitivity of yeast growth toward high ammonium concentrations and a dramatic increase in current amplitudes of rubidium and ammonium ions relative to K+ by 39-72-fold. These single site mutations gave rise to Rb+- and NH4/+ selective channels with Rb+ and NH4/+ currents that were approximately 10-13-fold greater in amplitude than K+ currents, whereas the NH+ to K+ current amplitude ratio of wild type KAT1 was 0.28. This strong conversion in cation specificity without loss of general selectivity exceeds those reported for other mutations in the pore domain of voltage-dependent K+ channels. Yeast growth was greatly impaired by sodium in two other mutants at this site (T256E and T256Q), which were blocked by millimolar sodium (K( 1/2 ) = 1.1 mM for T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative K+ channel mutant selection in yeast. These data suggest that the size of the side chain of the amino acid at position 256 in KAT1 is important for enabling cation permeation and that this site plays a crucial role in determining the cation selectivity of hyperpolarization- activated potassium channels.