In the heart, Ca2+/calmodulin-dependent protein kinase II is critically involved in the regulation of Ca2+ homeostasis. Previously the predominant expression of a subclass of Ca2+/calmodulin-dependent protein kinase II delta-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 delta-subunit subclass, delta 2, delta 3, delta 4, and delta 9 in the developing heart of the rat. By semiquantitative RT-PCR isoform delta 3 was shown to be typically expressed in the heart, whereas delta 4 was expressed in skeletal muscle of adult rat. From embryonic day 14 up to the adult state of rat ventricular muscle, amounts of delta 9 transcripts remained unchanged, transcript levels of isoforms delta 2 and delta 3 were significantly increased, whereas level of delta 4 transcript was significantly decreased. Immunoblotting, using an antibody recognizing specifically those delta-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 delta 4, was dramatically diminished, whereas a significant increase in the signal at about 58 kDa was assumed to represent an increase in isoform delta 3. The protein signal at about 56 kDa, close to the predicted molecular mass of isoform delta 2, was high in the embryonic heart and significantly decreased after birth. Our data suggest the predominant expression of isoform delta 2 in the embryonic heart, establish delta 3 to be the typical isoform in the adult heart and define the skeletal muscle form delta 4 to be characteristic for fetal and neonatal stages of the heart.