The beta3 subunit of high-voltage-gated calcium channels is a peripheral membrane protein that copurifies with neural N-type calcium channels. Murine genomic clones containing the full coding sequence of beta3 were isolated and the exons were mapped and sequenced. The murine calcium channel beta3 subunit is encoded by a unique gene composed of 13 translated exons that spread over approximately 8 kb of genomic sequence. Alternatively spliced transcripts of the beta3 gene were identified and characterized. The primary structure of beta3 is highly conserved between the murine, human, rabbit and rat proteins (98% identity). The intron placement within that primary structure correlates with the previously postulated exon positions in transcripts encoding the members of the calcium channel beta subunit family and confirm a close evolutionary relationship of the beta3, beta1, beta2 and beta4 subunit genes.