Cav1.2 (alpha(1C)) and Cav1.3 (alpha(1D)) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate alpha(1D) from alpha(1C) Ca Currents (ICa-1.) in cardiomyocytes. Here, we established a physiological model to study alpha(1D) ICa-1 ill native myocytes using RNA interference. Transfection of rat neonatal carcdiomyocytes (RNC) with alpha(1C) 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 alpha(1C) gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total ICa-1 was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the carcdiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of alpha(1C) and alpha(1D) Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings. 2009 Published by Elsevier Inc.