Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study was to evaluate the effect of methylene blue in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, methylene blue inhibited angiogenesis in a concentration-dependent manner. In addition, when methylene blue was combined with sodium nitroprusside, a spontaneous generator of nitric oxide, an inhibition of angiogenesis was evident which was comparable with that observed by the application of methylene blue alone. Sodium nitroprusside, alone, caused a significant inhibition in basal angiogenesis. These results provide evidence that methylene blue inhibits angiogenesis independently of nitric oxide pathway and suggest that methylene blue may be useful for treating angiogenesis-dependent human diseases.