We have identified, expressed and characterized two new isoforms of the multifunctional Ca²⁺/calmodulin-dependent kinase (CaM kinase) cloned from rat heart. Both isoforms are variants of the neuronal δ-CaM kinase (termed δ_A-CaM kinase), and are designated as δ_B-CaM kinase and δ_C-CaM kinase. The new isoforms differ from δ_A-CaM kinase in its isoform-specific insert region, between nucleotides 984 to 1087 of the δ_A-CaM kinase cDNA. Replacing these 102 nucleotides, a sequence of 33 nucleotides which code for 11 amino acids (KRKSSSSQMM) are introduced in δ_B-CaM kinase. The δ_C-CaM kinase lacks all 102 nucleotides and the corresponding 34 amino acids which they encode. The predicted molecular masses of the δ_B- and δ_C-CaM kinase isoforms are 57697 Da and 56446 Da, respectively. Recombinant δ-CaM kinases purified from transfected COS-7 cells were found to associate into a larger holoenzyme estimated to contain 8 to 10 subunits. The relative subunit molecular masses on SDS-polyacrylamide gel electrophoresis are 59 kDa, 54 kDa and 52 kDa for δ_A-, δ_B- and δ_C-CaM kinase, respectively. All three isoforms showed a strict dependence on Ca²⁺/calmodulin for activity and exhibited the Ca²⁺-dependent autophosphorylation and resultant conversion to Ca²⁺-independent kinase activity, characteristic features of multifunctional CaM kinase. Phosphopeptide analysis after partial CNBr digestion suggests that δ_B-CaM kinase is the predominant soluble CaM kinase species purified from rat heart.