The novel Cav1.3 (α1D) L-type Ca²⁺ channel plays a significant role in sinoatrial (SA) and atrioventricular (AV) nodes function and in atrial fibrillation. However, the characterization of α1D Ca²⁺ channel during heart development is very limited. We used real-time RT-PCR, Western blotting, and indirect immunostaining to characterize the developmental expression and localization of α1D Ca²⁺ channel in rat hearts. Both protein and mRNA levels of α1D Ca²⁺ channel decreased postnatally. Two forms of α1D Ca²⁺ channel protein (250 and 190 kD) were observed, with the full-length (250 kD) channel protein being predominant in the prenatal stages. Both Western blots and confocal imaging demonstrated that α1D Ca²⁺ channel protein was expressed in both atria and ventricles at fetal and neonatal stages but was absent in the adult ventricles. Interestingly, α1D Ca²⁺ channel was also found at the nucleus/perinucleus of immature but not adult atrial cells. Furthermore, the nuclear staining was reproduced in adult atrial cell line, HL-1 cells, which possess immature properties. The data are first to show that α1D Ca²⁺ channel has unique age-dependent expression profile and subcellular localization in the heart, suggesting a developmental stage-dependent specific function.