The endothelial cell receptor-tyrosine kinases, VEGF receptor 2 (VEGF-R2) and Tie-2, and their ligands, vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2, respectively, play key roles in tumor angiogenesis. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects on tumor growth. In this study, a human melanoma xenograft model (M21) was used to analyze the effects of simultaneous intradiabody depletion of vascular endothelial growth receptor-R2 and Tie-2 on tumor angiogenesis and tumor xenograft growth. The intradiabodies were expressed from recombinant adenovirus delivered through subtumoral injection. Blockade of both VEGF-R2 and Tie-2 pathways simultaneously or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2% and 74.4%, respectively). In addition, immunohistochemical staining of intradiabody-treated tumors demonstrated a decreased number of tumor-associated blood vessels versus control treatment. Previous studies with intrabodies had demonstrated that the Tie-2 receptor pathway was essential for tumor growth. The simultaneous blockade of the VEGF and Tie-2 pathways resulted in effective inhibition of tumor growth and demonstrated the potential of simultaneous targeting of multiple pathways as a therapeutic strategy.