Phospholamban is the regulator of the Ca²⁺‐ATPase in cardiac sarcoplasmic reticulum (SR). It is phosphorylated by a Ca²⁺/calmodulin‐dependent protein kinase (SRCaM kinase) which is closely associated with cardiac SR membrane preparations. We found that, upon renaturation of pig cardiac SR proteins, blotted onto PVDF membrane, two polypeptides of 54 and 52 kDa showed Ca²⁺/calmodulin‐dependent autophosphorylation. In Western blots of SR proteins, the 54/52 kDa polypeptides were recognized by an antibody specific for the δ‐CaM kinase isoforms, but not by an anti‐α‐CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti‐δ‐CaM kinase. The CaM kinase inhibitors KN‐62 and peptide CaMK‐(281–302) inhibited the activity of the SRCaM kinase with IC₅₀ values in the same range with those obtained for the brain isozyme. In addition, initial autophosphorylation (Ca²⁺‐dependent) produced a partially Ca²⁺‐independent enzyme while further autophosphorylation (Ca²⁺‐independent) made the enzyme completely Ca²⁺‐independent. Based on these results we suggest that the SRCaM kinase is a distinct δ‐CaM kinase isozyme.