Abstract: This study aimed to measure the concentration of intracytoplasmic free-Ca2+ in the in vitro matured bovine oocytes at MII stage and in the in vitro fertilized embryos at early different developmental stages 0 h (fertilization), 2 and 15 h (pronucleus stage) and 48 h (2-cell stage) after fertilization using Fura-2/AM as a Ca2+ fluorescent probe and to investigate Ca2+ spatial distribution in the intracytoplasm of bovine eggs at MII stage and 0, 2, 15 and 48 h after fertilization using Ca2+ fluorescent probe Fluo-3/AM. Meanwhile, the fertilized eggs were cultured in Ca2+-free culture medium to examine the effects of extracellular Ca2+ on the early embryo development. The results indicated that the intracellular Ca2+ concentrations of the bovine oocytes at MII stage and 0, 2, 15 and 48 h after fertilization were 84.99 nmol L-1 and 120.05, 229.09, 108.73 and 111.17 nmol L-1, respectively. No pronuleus or cleft eggs were found when cultured in Ca2+-free culture medium. These results suggested that the fertilization induced intracytoplasmic free-Ca2+ increase that pronucleus formation and egg cleavage were Ca2+ dependent that extracellular Ca2+ performed a specific function for the initiation of zygote development and that comparatively, a high level of Ca2+ concentrations was still maintained after pronucleus formation. Moreover, the spatial profile of fluorescence expression illustrated that a low density free-Ca2+ were homogenously distributed in the cytoplasm of oocytes at MII stage, the Ca2+ were concentrated at the cortex zone under the egg plasma membrane at fertilization, the Ca2+ intensity enhanced and spread towards the center of egg from edge at 2 h after fertilization and the Ca2+ were distributed in whole cytoplasm at pronucleus and first cleavage stage. These results demonstrated that extracellular Ca2+ is essential for normal embryonic development and also, confirm that Ca2+ increase is required for bovine oocyte activation at fertilization.