We compared effects of vascular endothelial growth factor-121 (VEGF¹²¹) and vascular endothelial growth factor-165 (VEGF¹⁶⁵) on generation of NO in HUVEC and the involvement of NO in VEGF¹²¹- and VEGF¹⁶⁵-induced angiogenesis. VEGF stimulated synthesis of NO within seconds, reaching peak concentrations of 450±25 and 180±15 nmol/l for VEGF¹²¹ and VEGF¹⁶⁵, respectively. The VEGF¹²¹ increased NO production for about 40 s while VEGF¹⁶⁵-stimulated NO release lasted only for about 20 s. Accordingly, cGMP elevation was stronger in VEGF¹²¹- than in VEGF¹⁶⁵-treated cells. The VEGF¹²¹ was a very weak mitogen but strong chemoattractant for HUVEC, whereas VEGF¹⁶⁵ potently induced both cell proliferation and migration. NO appeared to be involved in the endothelial migration and morphogenesis but not in the proliferation. NO was also a permissive molecule for VEGF¹²¹- but not for VEGF¹⁶⁵-induced capillary sprouting in spheroid culture. In conclusion, VEGF¹²¹ is a stronger stimulator of endothelial nitric oxide synthase (eNOS) activity, and angiogenic potential of VEGF¹²¹ is more reliant on NO contribution.