Genomic and cDNA clones encoding a novel Shaw-related potassium channel gene have been isolated from mice and humans. The mouse-Kv3.3 gene encodes a protein of 679 amino acids. Unlike the vertebrate Shaker-related genes that have intronless coding regions, mouse Kv3.3 is encoded by at least two exons separated by 3 kb of intervening sequence. The amino-terminal 212 amino acids are encoded by a single exon, and the hydrophobic core of the protein beginning at the S1 transmembrane segment is contained in a separate exon. Multiple Kv3.3-hybridizing transcripts are visible in the mouse brain, liver, thymus, and heart. Using probes derived from a human genomic clone containing the 3' exon of human Kv3.3 (KCNC3), we have localized the gene to human chromosome 19. The related gene, human Kv3.4 (KCNC4), was localized to human chromosome 1.