We report that the first localized Ca2+ transient visualized in the blastodisc cortex of post-mitotic zebrafish zygotes has unique features. We confirm that this initial ‘furrow positioning’ Ca2+ transient precedes the physical appearance of the first cleavage furrow at the blastodisc surface and that it has unique dynamics, which distinguish it from the subsequent furrow propagation transients that develop from it. This initial transient displays a distinct rising phase that peaks prior to the initiation of the two linear, subsurface, self-propagating Ca2+ waves that constitute the subsequent furrow propagation transient. Through the carefully timed introduction of the Ca2+ buffer, dibromo-BAPTA, we also demonstrate the absolute requirement of this initial rising phase Ca2+ transient in positioning the furrow at the blastodisc surface: no rising phase transient, no cleavage furrow. Likewise, the introduction of the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-aminoethoxydiphenyl borate, eliminates both the rising phase transient and the appearance of the furrow at the cell surface. On the other hand, antagonists of the ryanodine receptor and NAADP-sensitive channels, or simply bathing the zygote in Ca2+-free medium, have no effect on the generation of the rising phase positioning transient or the appearance of the furrow at the surface. This suggests that like the subsequent propagation and deepening/zipping Ca2+ transients, the rising phase furrow positioning transient is also generated specifically by Ca2+ released via IP3Rs. We propose, however, that despite being generated by a similar Ca2+ release mechanism, the unique features of this initial transient suggest that it might be a distinct signal with a specific function associated with positioning the cleavage furrow at the blastodisc surface.