Inositol 1,4,5-trisphosphate (IP₃) is one of the most important intracellular second messengers and is involved in elementary cellular functions. Modulation of cytosolic IP₃ levels is technically challenging and has spatio-temporal limitations. Airan et al. (Nature 458:1025-9) showed that IP₃ levels could be increased by using an optogenetic approach with a chimeric 7-transmebrane helix receptor consisting of rhodopsin and the a₁-adrenoreceptor (optoa₁-AR). This enables light-stimulation of the G_q signaling cascade and production of IP₃. Our long term goal is to use this tool for the analysis of IP₃ function during development and differentiation and for this purpose we generated a stable embryonic stem cell (ESC) line expressing optoa₁-AR fused to EYFP under control of the ubiquitous chicken b-actin promoter. Undifferentiated ESCs showed membrane bound EYFP fluorescence, and light-induced activation of the optoa₁-AR (470 nm, ~8 mW/mm²) caused an increase of cytosolic Ca²⁺. Furthermore, we investigated the role of IP₃ in cardiac pacemaking by differentiating cardiomyocytes from optoa₁-AR ESCs and by generating an optoa₁-AR transgenic HL-1 cardiomyocyte cell line. The cardiomyocytes were EYFP positive and specific illumination (470 nm, 4–17 mW/mm²) increased the frequency of spontaneous Ca²⁺ transients and diastolic Ca²⁺ levels. This was not observed in EGFP-expressing control cardiomyocytes and in optoa₁-AR HL-1 cells treated with the IP₃-receptor blocker 2-aminoethoxydiphenylborate or the phospholipase C blocker U-73122. We conclude that optoa₁-AR can be used to stimulate IP₃ signaling in ESCs and cardiomyocytes, thus providing a powerful tool for analyzing the spatio-temporal role of IP₃ in ES cell differentiation and cardiac pacemaking.