Cav1.2 (alpha1C) and Cav1.3 (alpha1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate alpha1D from alpha1C Ca currents (I(Ca-L)) in cardiomyocytes. Here, we established a physiological model to study alpha1D I(Ca-L) in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with alpha1C specific siRNA resulted in low silencing efficiency (50-60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the alpha1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total I(Ca-L) was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of alpha1C and alpha1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.