Purpose: A number of cytotoxic chemotherapy agents tested at low concentrations show antiangiogenic properties with limited cytotoxicity, e.g., cyclophosphamide, tirapazamine, and mitoxantrone. AQ4N is a bioreductive alkylaminoanthraquinone that is cytotoxic when reduced to AQ4; hence, it can be used to target hypoxic tumor cells. AQ4N is structurally similar to mitoxantrone and was evaluated for antiangiogenic properties without the need for bioreduction.@ Experimental Design: The effect of AQ4N and fumagillin on human microvascular endothelial cells (HMEC-1) was measured using a variety of in vitro assays, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound scrape, tubule formation, rat aortic ring, and invasion assays. Low-doseAQ4N (20 mg/kg) was also given in vivo to mice bearing a tumor in a dorsal skin flap.@ Results: AQ4N (10(-11) to 10(-5) mol/L) had no effect on HMEC-1 viability. AQ4N (10(-9) to 10(-5) mol/L) caused a sigmoidal dose-dependent inhibition of endothelial cell migration in the wound scrape model. Fumagillin showed a similar response over a lower dose range (10(-13) to 10(-9) mol/L); however, the maximal inhibition was less (25% versus 43% for AQ4N). AQ4N inhibited HMEC-1 cell contacts on Matrigel (10(-8) to 10(-5) mol/L), HMEC-1 cell invasion, and sprouting in rat aorta explants. Immunofluorescence staining with tubulin, vimentim, dynein, and phalloidin revealed that AQ4N caused disruption to the cell cytoskeleton. When AQ4N (20 mg/kg) was given in vivo for 5 days, microvessels disappeared in LNCaP tumors grown in a dorsal skin flap.@ Conclusions: This combination of assays has shown that AQ4N possesses antiangiogenic effects in normoxic conditions, which could potentially contribute to antitumor activity.