The expressed sequence tag (EST) database is a valuable tool to identify functionally related clones, when sequence similarity is so low that standard library screening methods cannot be successfully applied. Comparing conserved protein sequences of cloned voltage-gated potassium channels led to the identification and cloning of a new putative potassium channel alpha-subunit from rat brain, Kv9.1. A homologue of this alpha-subunit was also found in human brain tissue. A second alpha-subunit, Kv9.3, most probably belonging to the same subfamily, was also isolated and sequenced. Tissue distribution and analysis of genomic DNA were performed for both channels. rKv9.1 is expressed exclusively in the central nervous system, whereas rKv9.3 shows a widespread tissue distribution. No currents could be measured in X. oocytes upon injection of rKv9.1 or rKv9.3 cRNA.