Calsequestrin (CS) is a Ca(2+)-storing protein present in the sarcoplasmic reticulum (SR) of muscle cells. Calreticulin (CR) is a functional homologue of CS in the endoplasmic reticulum (ER) of non-muscle cells. During skeletal muscle differentiation, the major Ca(2+)-storing protein switches from CR to CS. To study the regulation of CS and CR expression in cardiomyocytes and the morphological maturation of Ca(2+)-storing sites in the SR, we examined rat hearts at various developmental stages and cultured adult cardiomyocytes by Western blotting and immunocytochemical analyses. CR was expressed in 14-day-old fetal rat cardiomyocytes, but disappeared gradually by 1 week after birth. CR reappeared in dedifferentiated adult cardiomyocytes in long-term culture. On Western blots, the concentration of CS in the heart did not change during development. Immunostaining for CS in fetal or neonatal rat cardiomyocytes revealed as scattered dots. CS-positive structures increased with development, and the regular striated distribution of CS at Z lines was completed around 3 weeks after birth. These results indicated that (1) CR expression is downregulated during cardiac differentiation and upregulated during dedifferentiation, and (2) maturation of SR involves the organization of CS-positive structure after birth.