Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to participate in signal transduction. In vertebrates, five different calpains have so far been identified, of which three, mu-, m-, and mu/m-calpain, are ubiquitously expressed while the other two, nCL-1 (p94) and nCL-2, exhibit a restricted tissue distribution. We have identified two new vertebrate calpain genes, Capn5 and Capn6. The human and mouse amino acid sequences of these new calpains are the most divergent of the vertebrate calpains identified. They possess most of the residues conserved in calpain family members but the C-terminal region lacks any homology to the calmodulin-like domain of other vertebrate calpains. They both exhibit significant homology over the entire coding region to the protein encoded by the gene tra-3, involved in nematode sex determination, and Capn5 may represent its vertebrate orthologue. The predicted Capn6 protein lacks critical active site residues and may not be proteolytically active. Both genes are differentially expressed in human tissues with highest RNA levels for Capn5 occurring in the testis, liver, trachea, colon, and kidney, while Capn6 is highly expressed only in the placenta sample of the 50 tissues examined. Phylogenetic analysis suggests that the vertebrate calpains arose through a series of gene duplication events that began before the initial divergence of the vertebrate and invertebrate lineages. The discovery of these two new calpains highlights a hitherto unknown complexity of the calpain family with subclasses perhaps possessing different modes of regulation.