In this study, we report the cloning of the rat cardiac isoform of calsequestrin on the basis of its interaction with an epsilonprotein kinase C-unique sequence (epsilonV1) derived form the epsilonprotein kinase C regulatory domain. Calsequestrin binds activated epsilonprotein kinase C holoenzyme better than the inactive enzyme and nearly three times better than other protein kinase C isozymes. The interaction between epsilonprotein kinase C and calsequestrin is mediated by sequences in both the regulatory and kinase domains of the epsilonprotein kinase C. Finally, we show that calsequestrin is an epsilonprotein kinase C substrate in vitro and protein kinase C phosphorylation of calsequestrin leads to a decreased binding of epsilonprotein kinase C to calsequestrin.