Hydrogen peroxide (H(2)O(2)) has been implicated as a key molecule in arresting embryonic development; however, its mechanism of action is not fully established. The aim of the present study was to determine the chronological generation of H(2)O(2) from oocyte to morula, and to examine the relationship of H(2)O(2) with loss of mitochondrial membrane potential, nuclear factor kappa-B (NF-kappaB), p53, caspase-3 activation, and cell death in bovine embryos in vitro. Accordingly, superoxide anion radicals were detected between 32 and 120 h after in vitro fertilization, but higher percentages of oxygen radicals were found in non-competent embryos (n=73, 22 to 34%) than in competent embryos (n=73, 0 to 1%; P<0.005). Similarly, H(2)O(2) levels were higher in non-competent embryos (n=249, 39 to 71%) than in competent embryos (n=278, 0 to 3.4%) at all developmental stages tested (P<0.005). The percentage of cells with apoptotic morphology were higher in non-competent embryos (n=411, 3 to 54%) than in competent embryos (n=306, 0 to 0.6%; P<0.005). Based on assessment of mitochondrial membrane potential, competent embryos (n=305) had the highest percentages of JC-1 staining (31 to 50%) when compared with non-competent embryos (n=411; 1 to 15%, P<0.005). The percentage of activation of general caspases was different in non-competent embryos (n=291, 15 to 57%) when compared to competent embryos (n=304, 0 to 0.5%; P<0.005). Pharmacological inhibition of caspase-3, NF-kappaB and p53 triggered aberrant embryo cytoplasmic fragmentation with and without nuclei. We concluded that the sequential mechanism of O(2)(-) and H(2)O(2) generation, mitochondrial damage, caspase activation, and apoptotic morphology might be responsible for the developmental arrest of preimplantation embryos.