Background: Two types of L-type Ca Channels are expressed in the heart: Cav1.2 (α1C) and Cav1.3 (α1D). In contrast to α1C, α1D Ca channel is highly expressed in the sinoatrial node and atria, and is involved in the impulse generation and propagation through the AV node. Deletion of the α1C gene results in embryonic lethality before E14.5 and there are no pharmacological or biophysical means to separate α1D from α1C Ca currents. The aim of this study was to establish a model to characterize α1D Ca current using the novel technology of RNA interference.Methods and Results: Rat neonatal cardiomyocytes (RNC) were transfected with α1C specific siRNA using lipofectamine which resulted in 50% silencing at the mRNA/protein level. Limited by the low transfection effieciency (50%) in the RNC, we cotransfected α1C siRNA with cy3 labeled Human GAPDH siRNA and sorted out the fluorescent cells using FACS to separate tranfected and non-transfected cells. The level of silencing of α1C in enriched transfected cells reached only 65% efficiency. To achieve higher transfection efficiency, we generated and infected RNC with a lentivirus construct carrying the α1C siRNA sequence under a U6 promoter. Using this model, we achieved 100% transfection efficiency, and more than 90% silencing of the α1C gene confirmed by real-time PCR, Western blot, and immunoflourecence. These biochemical results were confirmed electrophysiologically by measurements of total L-type Ca current which was reduced by 80% in transfected cells.Conclusion: Lentiviral shRNA is an efficient model for post-transcriptional gene silencing of ion channels in primary cardiomyocytes. This novel approach provides a valuable mean for assessing the differential roles of α1C and α1D Ca channels in native cardiomyocytes and could be used to examine their roles in physiological and pathological settings.