Alzheimer's disease is associated to a cerebral amyloid angiopathy with dysregulation of cerebral blood flow (CBF). In vitro studies have shown that short-term application of β-amyloid (Aβ) peptides to isolated vessels affects vascular tone within 1h, but no studies have examined the effect of long-term incubation with Aβ. Here we evaluate the effect of Aβ_(1–40) and Aβ_(25–35) in rat basilar artery for up to 24h. Basilar artery segments were incubated with 25μM Aβ_(1–40) or Aβ_(25–35), for 6 or 24h. After treatment, arteries were mounted in a wire myograph, in physiological salt solution gassed with O₂/CO₂, in the absence of Aβ, and challenged with vasoconstrictors and vasodilators. Vasomotor responses were not significantly changed by 6h treatment with Aβ peptides whereas 24h treatment with either Aβ_(25–35) or Aβ_(1–40) increased vasoconstriction to 5-hydroxytryptamine (5-HT) and reduced endothelium-dependent vasodilatation to acetylcholine (ACh). Analysis of endothelial cells did not show apoptotic changes associated to endothelial dysfunction, as assessed by TUNEL immunostaining and examination of nuclear morphology, but basal phosphorylation of endothelial nitric oxide synthase (at serine 1177) appeared reduced. These data suggest that long incubation with Aβ peptides induces an alteration of endothelial function in isolated basilar artery, involving eNOS activity without changing cell morphology. This endothelial dysfunction may play a role in the pathogenesis of CBF dysregulation occurring in cerebral amyloid angiopathy and Alzheimer's disease.