Ca²⁺ is a ubiquitous second messenger which plays a key role in early development of embryos. Ca²⁺ probes (Fluo-4 or Indo-1) were injected into zebrafish eggs to detect the distribution of free Ca²⁺ during their first cleavage using confocal microscopic or dual-wavelength ratiometric imaging. A high Ca²⁺ zone was first observed in the animal pole right before the first cleavage, then it extended along the cleavage furrow and the Ca²⁺ signal remained high in this region throughout the first cleavage. Intracellular Ca²⁺ concentration ([Ca]_i) was measured via Indo-1 dual-wavelength system, and it was shown to be homogeneous within the whole embryo before the first cleavage. During the first cleavage, [Ca]_i increased significantly near the cleavage furrow, while it remained unchanged in other areas. As the dual-wavelength ratiometric imaging eliminates the artifacts due to indicator inhomogeneity, the results provided an unequivocal quantification for the Ca²⁺ dynamics associated with the first cleavage of embryonic development.