As an alternative to electronic pacemakers, we explored the feasibility of converting ventricular myocytes into pacemakers by somatic cell fusion. The idea is to create chemically induced fusion between myocytes and syngeneic fibroblasts engineered to express HCN1 pacemaker channels (HCN1-fibroblasts). HCN1-fibroblasts were fused with freshly isolated guinea pig ventricular myocytes using polyethylene-glycol 1500. In vivo fused myocyte-HCN1-fibroblast cells exhibited spontaneously oscillating action potentials; the firing frequency increased with beta-adrenergic stimulation. The heterokaryons created ectopic ventricular pacemaker activity in vivo at the site of cell injection. Coculture of nonfused HCN1-fibroblasts and myocytes without polyethylene-glycol 1500 revealed no evidence of dye transfer, demonstrating that the I(f)-mediated pacemaker activity arises from heterokaryons rather than electrotonic coupling. This nonviral, non-stem cell approach enables autologous, adult somatic cell therapy to create biopacemakers.