A biological pacemaker might be created by generation of a cellular construct consisting of cardiac cells that display spontaneous membrane depolarization, and that are electrotonically coupled to surrounding myocardial cells by means of gap junctions. Depending on the frequency of the spontaneously beating cells, frequency regulation might be required. We hypothesized that application of Kir2.1 expressing non-cardiac cells, which provide I (K1) to spontaneously active neonatal cardiomyocytes (NCMs) by electrotonic coupling in such a cellular construct, would generate an opportunity for pacemaker frequency control. Non-cardiac Kir2.1 expressing cells were co-cultured with spontaneously active rat NCMs. Electrotonic coupling between the two cell types resulted in hyperpolarization of the cardiomyocyte membrane potential and silencing of spontaneous activity. Either blocking of gap-junctional communication by halothane or inhibition of I (K1) by BaCl(2) restored the original membrane potential and spontaneous activity of the NCMs. Our results demonstrate the power of electrotonic coupling for the application of specific ion currents into an engineered cellular construct such as a biological pacemaker.