Combretastatin-A4-phospate (CA4P) is currently in clinical trials for cancer therapy. The active derivative CA4 binds to tubulin in endothelial cells and destabilizes the cytoskeleton, leading to vascular damage and tumour hypoxia. However, in 35% of the patients hypertension has been reported. The hypothesis of this study was that CA4P by destabilization of the cytoskeleton followed by decreased Src tyrosine kinase activity leads to less NO and hypertension. Rat mesenteric and femoral small arteries were isolated and mounted in microvascular myographs for functional studies. Human umbilical vein endothelial cells (HUVEC) were used to explore the pathways affected by CA4P. In femoral arteries phenylephrine concentration-response-curves were leftward shifted in the presence of a NO synthase inhibitor, L-NOARG, and by CA4P, but not by the dephosphorylated form, CA4. In mesenteric arteries there was no difference in phenylephrine contraction in the presence or absence of CA4P and CA4. Electric field stimulation of the arteries showed that CA4P enhanced vasoconstriction in femoral arteries at low frequencies, while this was not observed in mesenteric arteries. In mesenteric arteries vanadate induced vasorelaxation which was endothelium-dependent and reduced by CA4P and L- NOARG. CA4P did not change acetylcholine- or flow-evoked vasodilatation. Vanadate-induced phosphorylation of serine 1177 in endothelial NO synthase was blunted in the presence of CA4P, but not by CA4. The present findings suggest that CA4P enhances sympathetic adrenergic vasoconstriction by blocking NO synthase, possibly through inhibition of Src kinase. This mechanism is tissue specific, and it may contribute to the hypertensive side-effects of CA4P in cancer patients.