The extent to which mitogenic growth factors influence embryo development is not well characterized. We sought to determine the effect of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on naturally fertilized (in vivo-derived) and in vitro-fertilized mouse embryos, compared with that on cloned (intracytoplasmic nuclear injection-derived) mouse embryos, in which EGF and TGFalpha expression is markedly reduced. Immunoneutralization of EGF, TGFalpha, and EGF receptor by using specific antibodies significantly reduced the blastocyst development rate (in vivo-derived: 66%, 63%, and 63%, respectively; in vitro-fertilized: 57%, 55%, and 56%, respectively), increased the number of apoptotic nuclei (in vivo-derived: 9%, 10%, and 9%, respectively; in vitro-fertilized: 13%, 13%, and 13%, respectively), decreased the total number of cells (in vivo-derived: 87%, 85%, and 86%, respectively; in vitro-fertilized: 86%, 85%, and 86%, respectively), and increased the inner cell mass:trophectoderm ratios (in vivo-derived: 1:2.70 +/- 0.05, 1:2.73 +/- 0.04, 1:2.71 +/- 0.06, respectively; in vitro-fertilized: 1:2.94 +/- 0.02, 1:2.96 +/- 0.02, 1:2.95 +/- 0.02, respectively). In most cases, combined treatment with neutralizing antibodies to both EGF and TGFalpha accentuated changes in these parameters. Further, the effect of combined immunoneutralization on these parameters in fertilized embryos was no different from those in cloned embryos. Therefore, normal expression of mitogenic growth factors is crucial for successful development of mouse embryos before implantation. Inhibiting the action of mitogenic growth factors causes fertilized embryos to exhibit developmental characteristics similar to those of cloned embryos, which may partially explain the poor developmental potential of cloned mammalian embryos.