Calpains are Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both are heterodimers, consisting of a distinct catalytic subunit and a common regulatory subunit. We describe cloning and sequencing of the calpain small (regulatory) subunit (cpns) cDNA from rainbow trout. This represents the first fish and lower vertebrate full cDNA of cpns. The rainbow trout cpns cDNA was used to retrieve the zebra fish and Japanese flounder homologues. We present evidence that fish cpns, unlike the conventional mammalian predominant isoform, cpnsl, is lacking the glycine-rich region of domain V. Because the glycine-rich region is known to play a role in membrane targeting, this divergent cpns suggests potentially different functional and activation mechanisms of the fish calpain system. A phylogenetic tree for the cpns gene superfamily has been constructed and the evolution of cpns considered.