Optogenetics has become a powerful tool in neuroscience either as sensors to survey the function at the cellular level or as actuators to regulate the neuronal activity. The calyx-type presynaptic terminal of embryonic chick ciliary ganglion (CG) is relatively large and therefore accessible to physiological recording methods such as the patch clamp. As it innervates the postsynaptic ciliary neuron in a one-to-one manner at embryonic day 14 (E14), its physiology corresponds to the morphology and electrophysiological responses of its postsynaptic cell. Further progress can be expected if the presynaptic mechanisms of these large terminals can be studied using optogenetics. Here, we carried out simultaneous optogenetic probing and manipulation of this presynaptic terminal and evaluated optogenetics for the presynaptic research. We transfected the plasmid vectors containing one of chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and a red-shifted fluorescent Ca2+-sensor, R-GECO1, into the midbrain preganglionic neurons at E2 using in ovo electroporation technique (Odani et al., 2008). At E14, the presynaptic terminals in the ciliary ganglion were used for the physiological experiments under high-speed confocal microscopy. The presynaptic Ca2+ elevation was triggered by a direct photostimulation of the presynaptic terminal via optical fiber. Although this optically-evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the presence of TTX and N-type voltage-dependent channel inhibitor, ω-conotoxin GVIA, or in the absence of extracellular Ca2+. The remaining component was depleted by repetitive stimulations with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, thapsigargin. These results suggest that the photo-activation of ChRFR facilitated the Ca2+ release from endoplasmic reticulum Ca2+ stores directly or indirectly. Therefore, it is necessary to keep in mind that such background Ca2+ elevation could modify the biochemical milieu regulating the transmitter release.