In the heart, Ca 2ϩ /calmodulin-dependent protein kinase II is critically involved in the regulation of Ca 2ϩ homeostasis. Previously the predominant expression of a subclass of Ca 2ϩ /calmodulin-dependent protein kinase II ␦-subunit, containing a second variable domain, was demonstrated in cardiac tissue. Here we report on the expression pattern of the non-neuronal members of this ␦-subunit subclass, ␦ 2 , ␦ 3 , ␦ 4 , and ␦ 9 in the developing heart of the rat. By semiquantitative RT-PCR isoform ␦ 3 was shown to be typically expressed in the heart, whereas ␦ 4 was expressed in skeletal muscle of adult rat. From embryonic day 14 up to the adult state of rat ventricular muscle, amounts of ␦ 9 transcripts remained unchanged, transcript levels of isoforms ␦ 2 and ␦ 3 were significantly increased, whereas level of ␦ 4 transcript was significantly decreased. Immunoblotting, using an antibody recognizing specifically those ␦-isoforms containing the second variable domain, revealed three separated protein signals at about 59 kDa, 58 kDa, and 56 kDa. The immunoreaction at about 59 kDa, corresponding to the predicted molecular mass of ␦ 4 , was dramatically diminished, whereas a significant increase in the signal at about 58 kDa was assumed to represent an increase in isoform ␦ 3 . The protein signal at about 56 kDa, close to the predicted molecular mass of isoform ␦ 2 , was high in the embryonic heart and significantly decreased after birth. Our data suggest the predominant expression of isoform ␦ 2 in the embryonic heart, establish ␦ 3 to be the typical isoform in the adult heart and define the skeletal muscle form ␦ 4 to be characteristic for fetal and neonatal stages of the heart.