Electrical pacing of cells stimulates excitable cells by only evoking free-running action potentials and the area of stimulation is difficult to modulate. Herein we report the use of the light-gated cation channel channelrhodopsin 2 (ChR2) for sub-threshold depolarization, modulation and evocation of action potentials in cardiomyocytes in vitro and in vivo with high spatio-temporal resolution. ES cells were transfected with the ChR2-EYFP fusion protein under control of the chicken b-actin promoter. Both, ES cells and ES cell-derived cardiomyocytes showed membrane bound EYFP fluorescence. Application of light (450nm) evoked a non-selective current and ensuing depolarization. Constant application of low-intensity light enhanced spontaneous beating frequencies of cardiomyocytes, whereas brief (5ms) high-intensity light stimulation triggered free-running action potentials. Longer illumination led to prolonged action potentials and to an increase of absolute refractory periods. Monolayers of ChR2 expressing cardiomyocytes plated on multi-electrode-arrays could be locally stimulated by brief (5ms) and confined (0.3mm²) illumination whereas constant stimulation induced electrical uncoupling of illuminated areas. For light-induced stimulation of hearts, transgenic mice were generated using diploid complementation of transgenic ES cells. Brief (10ms) illumination of EYFP positive atrial or ventricular areas from chimeric mice led to extrabeats whereas illumination of negative areas had no effect. ECG-triggered illumination was used to determine the refractory period where no extrabeats could be evoked and the vulnerable phase where free running ventricular tachycardia could be induced. Constant illumination with low intensity light enhanced vulnerability with ventricular extrabeats occurring spontaneously. Further experiments with different spatio-temporal stimulation patterns will allow us to study mechanisms underlying the onset of arrhythmias in vitro and in vivo.