The gamma subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct gamma subunits have been described from human and/or mouse. The first identified member of this group of proteins, gamma(1), is a component of the L-type calcium channel expressed in skeletal muscle. A second member, gamma(2), identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel gamma subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian gamma subunits suggests the following relationship ((((gamma(2), gamma(3)), (gamma(4), gamma(8))), (gamma(5), gamma(7))), (gamma(1), gamma(6))) that indicates that they evolved from a common ancestral gamma subunit via gene duplication. Our analysis reveals that the novel gamma subunit gamma(6) most closely resembles gamma(1) and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other gamma subunits. Rat gamma subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of gamma(1) mRNA and the long isoform of gamma(6) mRNA is most robust in skeletal muscle, while gamma(6) is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the gamma subunits, we propose that gamma(1) and gamma(6) may share common physiological functions distinct from the other homologous gamma subunits.