A conservative reversion at position 374 in a chimeric K+ pore, CHM, switched the preferred ionic conductance from K+ to Rb+. To understand how selectivity was switched, codons for 18 different amino acids were substituted at position 374 in each of two different K+ channels CHM and Kv2.1, the host channel for CHM. After injection of cRNA into Xenopus oocytes, less than half of the substituted mutants expressed functional channels. In both CHM and Kv2.1, channels with the substituted hydrophobic residues Val or Ile expressed Rb+-preferring pores while channels with the substituted polar residues Thr or Ser expressed K+-preferring pores. Val or Ile stabilized while Thr or Ser destabilized blockade by internal tetraethylammonium (TEA) confirming the importance of hydrophobic interactions for blockade. TEA blockade was dependent upon the charge carrier and was more effective in the presence of the ion having the larger conductance. The results are consistent with a model in which the side chains at position 374 form a filter for K+ and Rb+ ions and a site for blockade by internal TEA.