Despite their important role in controlling the cardiac Ca²⁺ homeostasis, presence and functions of individual isoforms of the multifunctional Ca²⁺/calmodulin‐dependent protein kinase in the heart are not well studied. Here we report on expression of isoforms of the δ class in two differentiation states of the embryonic rat heart‐derived cell line H9c2 compared to adult rat heart. Reverse transcription coupled polymerase chain reaction analysis revealed specific expression patterns of four variants of the δ class (δ_B, δ_C, δ₄, δ₉) in adult rat heart, H9c2 myoblasts, and skeletal muscle‐like H9c2 myotubes. δ_C was identified as a common isoform with higher amounts in H9c2 cells and the prominent one in myoblasts. In contrast, expression of δ₉ accompanied cardiac as well as skeletal muscle differentiation. Expression of δ_B, however, was representative for differentiated cardiac muscle, whereas δ₄ expression coincided with differentiation into the skeletal muscle‐like state. Our results demonstrate differentiation‐dependent isoform expression of the δ class of the multifunctional Ca²⁺/calmodulin‐dependent protein kinase of muscle. The identification of cardiac target proteins for this kinase, e.g. the α₁‐subunit of the L‐type Ca²⁺ channel, the sarcoplasmic reticulum Ca²⁺‐ATPase, phospholamban and the ryanodine receptor define H9c2 myoblasts as a suitable model system for further functional characterization of the identified cardiac δ isoforms. J. Cell. Biochem. 68:259–268, 1998. © 1998 Wiley‐Liss, Inc.