Vertebrate embryos generate striking Ca2+ patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca2+ during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca2+ along the dorsal–ventral axis, with higher Ca2+ concentrations in the ventral margin and lower Ca2+ concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca2+ pump with 0.5μM thapsigargin elevates cytosolic Ca2+ in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left–right asymmetry. Brain defects include a left–right reversal of pitx2 expression in the dorsal diencephalon and a left–right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca2+ pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca2+ pump during gastrulation inhibits expression of no tail (ntl) and left–right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer’s vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca2+ plays a role in Kupffer’s vesicle function, influencing ciliary motility and translating the vesicle’s counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca2+ plays an additional role in the formation of Kupffer’s vesicle.