During early vertebrate development, a signaling network is activated along the midline of the embryo. This signaling network induces the neural tube floor plate and ventral brain regions. In turn, induction of the ventral brain region is important for bilateral division of the forebrain and bilateral separation of the eyes. The present study provides direct evidence for a role of the endoplasmic reticulum Ca(2+) pump in zebrafish midline signaling. The endoplasmic reticulum Ca(2+) pump was inhibited in zebrafish embryos using thapsigargin or cyclopiazonic acid. Inhibition of the endoplasmic reticulum Ca(2+) pump during early gastrulation induces cyclopia, mimicking defects observed in cyclops, squint, one-eyed pinhead, and silberblick mutant embryos. In contrast, inhibition of the endoplasmic reticulum Ca(2+) pump during mid-gastrulation does not induce cyclopia, but does induce tail defects, mimicking defects observed in no-tail mutant embryos. This study is the first to relate thapsigargin and cyclopiazonic acid with induction of cyclopia. In addition, obtained results provide new information on the roles of Ca(2+) in embryonic development and may lead to new insights on the mechanisms underlying holoprosencephaly, a relatively common brain defect in human development.