Purpose: Vascular endothelial growth factor (VEGF) is a potent angiogenic agent and plays a major role in tumor growth and metastases. We have previously reported the locoregional (i.p.) delivery of adenovirus-mediated antiangiogenic soluble FLT-1 (sFLT-1; a naturally encoded potent VEGF antagonist) gene therapy to inhibit VEGF action in a murine ovarian carcinoma model. This study was predicated on the fact that systemic delivery of sFLT-1 might allow an approach for therapy of disseminated tumor. The purpose of this study is to test the effects of i.v. delivered, adenovirus-mediated sFLT-1 on the survival duration in a murine ovarian tumor model and to evaluate the safety of i.v.-delivered versus i.p.-delivered adenovirus-mediated sFLT-1 in non-tumor-bearing mice. Experimental Design: To determine the effects of i.v.-administered adenovirus-mediated sFLT-1 on survival duration of mice bearing i.p. human ovarian tumors, an E1A/B-deleted, (replication-deficient) infectivity-enhanced recombinant adenovirus AdRGDGFPsFLT-1 encoding cDNA for both sFLT-1 and GFP (green fluorescent protein), a control adenovirus AdRGDGFP encoding GFP alone, or PBS was delivered i.v. The therapeutic effect of sFLT-1 was evaluated by survival duration of the mice. Furthermore, the safety of i.v.- or i.p.-delivered adenovirus-mediated sFLT-1 was evaluated by administering AdRGDGFPsFLT-1, AdRGDGFP, or PBS either i.v. or i.p. into non-tumor-bearing mice. Adenovirus-mediated gene expression was determined by determining GFP expression using fluorescent microscopy and by assessing sFLT-1 expression in liver, lungs, spleen, and kidneys by immunohistochemistry using anti-FLT-1 monoclonal antibody. Systemic levels of sFLT-1 were evaluated by ELISA and the toxicity was evaluated by histopathology. Results: The i.v. delivery of AdRGDGFPsFLT-1 in the ovarian tumor model resulted in a shorter duration of survival of the mice as compared with the control group. Furthermore, in the safety evaluation experiment, i.v. administration of AdRGDGFPsFLT-1 in non-tumor-bearing mice principally localized to the liver. This localization lead to sFLT-1 overexpression, mainly in the liver, resulting in hemorrhage and tissue toxicity. However, i.p. delivery of AdRGDGFPsFLT-1 did not localize principally to the liver, leading to negligible expression of sFLT-1, and no intrahepatic hemorrhage or toxicity was observed. The i.v. delivery of the control virus AdRGDGFP also principally localized to the liver, leading to GFP expression mainly in the liver. However, neither hemorrhage nor morphological cytotoxicity was observed, i.p. delivery of AdRGDGFP resulted in ectopic localization to the liver with very little GFP expression and no toxicity. These results suggest that overexpression of sFLT-1 in the liver as a result of i.v. delivery is hepatotoxic. Conclusions: Our results suggest that i.v. delivery of the sFLT-1 gene via replication-deficient, infectivity-enhanced recombinant adenoviral vectors will result in overexpression of sFLT-1 in the liver leading to unacceptable hepatotoxicity. Tumor-specific targeting of the vectors and tumor-specific expression strategies should be used to ensure a clinically useful antiangiogenesis gene therapy.