||Transgenic zebrafish that express apoaequorin (the protein part of bioluminescent Ca2+ reporter, aequorin) specifically in the musculature were generated to study muscle Ca2+ signals during late somitogenesis. Two distinct periods of spontaneous, synchronized Ca2+ signals, with characteristic frequencies and durations, termed ‘Signaling Period 1’ (SP1) and ‘Signaling Period 2’ (SP2), were visualized in the trunk of intact embryos between ~17.5-19.5 hours post fertilization (hpf) and after ~23 hpf, respectively, separated by a quiet period. The individual Ca2+ signals were further characterized with respect to their spatial signatures using the fluorescent Ca2+ reporter, calcium green-1 dextran (10 kDa) in conjunction with confocal microscopy. These experiments revealed that the signals appeared to be restricted to slow muscle cells (SMCs) and that the SP1 Ca2+ signals had both a significant nuclear as well as cytoplasmic component, whereas the SP2 signals were predominantly cytoplasmic. These signals had, therefore, characteristic frequencies, durations and locations within developing SMCs. Aequorin-based imaging of cyclopamine- or forskolin-treated embryos, and of smu-/- mutant embryos, in which SMCs do not form, confirmed the specific cellular location of the Ca2+ signals. Furthermore, treating embryos with antagonists of the nicotinic acetylcholine receptor (nAChR; i.e., α-bungarotoxin), the dihydropyridine receptor (DHPR; i.e., nifedipine), the inositol 1,4,5-trisphosphate receptor (IP3R; i.e., 2-APB) and the ryanodine receptor (RyR; i.e., ryanodine) suggested that the SP1 signals were mediated through endogenous neuronal activation of AChRs and then DHPRs to release Ca2+ from intracellular stores via both IP3Rs and RyRs. Immunohistochemistry showed that the differential expression and organization of IP3Rs and RyRs in SMCs coincided with the initial generation of the SP1 Ca2+ transients and their subsequent spatial nature. Furthermore, 2-APB and ryanodine were both shown to disrupt the organization of the SMCs, but in different ways. This suggests that different Ca2+ signaling pathways may be responsible for different functions during development of SMCs.