RNA interference (RNAi) by short double-stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Initially discovered in invertebrates and plants, RNAi has become the currently most widely utilized technique in functional genomic studies in mammalian cells. Whereas efficient gene silencing has been demonstrated in various cell lines and developing cells of embryonic origin, thus far successful application of this technique in adult cardiac myocytes, a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.1) in adult rod-shaped cross-striated rat ventricular myocytes in vitro using adenovirus-driven transcription of siRNA hairpins under murine U6 promoter control. EGFP fluorescence or density of background inward rectifier current (IK₁) were reduced by > 90 % within 6 to 8 days after transformation with the corresponding virus. SiRNA-mediated gene-silencing of these proteins was confirmed on the mRNA-level by real time RT-PCR. Knockdown of Kir2.1 resulted in depolarization and in a fraction of cells caused pacemaker activity.