RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), 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 ACM in vitro using adenovirus driven transcription of siRNA hairpins. EGFP fluorescence and density of background inward rectifier current (IK1) were reduced by >90% within about 6–8 days after transformation with the corresponding virus. In Kir2.1-silenced myocytes resting membrane potential was significantly reduced. Survival of these cells in culture was compromised, presumably due to Ca2+-overload caused by the depolarization. The sequence-specific knockdowns of EGFP and Kir2.1 were confirmed on the RNA level using real-time RT-PCR. In Kir2.1-silenced myocytes density of transient outward current, carried predominantly by Kv4.x subunits remained unaffected. This communication for the first time demonstrates proof of principle of efficient RNA interference using adenovirus-based vectors and demonstrates its large potential in phenotyping of ACM.