Numerous studies, performed mainly on dissociated cells, have shown that calcium signals have a role during different stages of neuronal development. However, the actions of calcium during neuronal development in vivo remain to be established. The present study has investigated the role of intracellular calcium signals during development of motoneurons in the spinal cord of intact zebrafish embryos. Loading blastomeres of early embryos with either the calcium buffer BAPTA or the calcium reporter dye Calcium Green, was shown to disrupt motoneuron development in the spinal cord of embryos at 24 h postfertilisation. Loading the calcium buffer BAPTA, at an intracellular concentration of 1 mM, into the blastomeres of early embryos did not alter the resting levels of intracellular calcium, but significantly dampened transient rises in intracellular calcium in the cells of later stage embryos. Loading cells with 1 mM BAPTA significantly decreased the number of motoneurons present in the spinal cord at 24 h, indicating that calcium signals are important for normal motoneuron differentiation. Furthermore, in those BAPTA-filled cells that did adopt a motoneuron cell fate, axogenesis was found to be inhibited, suggestive of a role for calcium signalling in neurite initiation. This work provides evidence that calcium signals are necessary at several stages of motoneuron development in vivo.