Tumor growth is accelerated by induction of angiogenesis regulated by many cytokines and growth factors. In a tumor mass, various angiogenic factors and their receptors are simultaneously expressed and the overlapped expressions of various factors may contribute to the aggressive growth of tumor. However, the possible combined effect and mechanism of growth factors involved in angiogenesis are still under investigated. Insulin-like growth factor-II (IGF-II) has been identified as an angiogenic factor and highly expressed in solid tumors. Here we demonstrated that another angiogenic factor, epidermal growth factor (EGF), synergistically induced the angiogenic activity when co-treated with IGF-II in vivo. We performed mouse Matrigel plug assay. Cotreatment of IGF-II and EGF resulted in a significant induction of functional new vessels in mouse Matrigel plug more than additive amount of vessels induced by each growth factor. However, synergism of these two factors was not found in in vitro angiogenic assays, i.e., in migration and proliferation assays. The metalloproteinase-2 (MMP-2) protein level was enhanced by co-treatment of IGF-II and EGF, similar to that of individual treatment of them or PMA or bFGF. EGF down-regulated hypoxia-induced IGF-II binding protein-3 (IGFBP-3), which may contribute to the enhancement of free IGF-II accessibility to its receptors in vivo. Moreover, the combination of IGF-II with EGF significantly induced bFGF mRNA level, a potent angiogenic molecule, which may also contribute to synergistic effect in vivo. These results suggest that IGF-II and EGF may synergistically cooperate to induce angiogenesis in vivo by indirect mechanisms, i.e., synergistic induction of another angiogenic factor and modulation of IGF-II's bioavailability.