A putative second potassium channel expressed in rat brain is identified by cloning and characterization of cDNA clones. Two cDNA clones, isolated from rat brain libraries, encode a 499-residue protein that is 80% identical with a previously described rat brain potassium channel and 68% identical with a Drosophila potassium channel. This new potassium channel is called BK2 to distinguish it from the previously described potassium channel (BK1). The BK2 gene, unlike the Drosophila potassium channel gene complex, appears to produce a single, large (approximately 9.5-kilobase) RNA transcript. Southern analysis of rat genomic DNA indicates that the BK1 and BK2 transcripts are the products of independent genes. Analysis of the distribution of BK2 transcripts in rat brain by in situ hybridization histochemistry suggests that the BK2 gene is ubiquitously expressed by central nervous system neurons. Identification of this putative second mammalian potassium channel cDNA indicates the existence of a potassium channel gene family, confirming electrophysiological data on the diversity of potassium channels expressed in rat brain.