The majority of cholinergic fibers in the cerebral cortex originate in basal forebrain nuclei. Fiber terminals from basal forebrain cholinergic areas have intimate contact not only with cortical neurons, but also with cortical parenchymal blood vessels, such as penetrating arteries and microvessels (1). Stimulation of basal forebrain cholinergic nuclei such as nucleus basalis of Meynert (NBM) produces an increase in cortical parenchymal blood flow, by activating muscarinic and nicotinic cholinergic receptors (2,3). An increase in cortical blood flow during NBM stimulation is uncoupled from cortical glucose metabolism, indicating that cholinergic projection from NBM is important for vascular control in the cerebral cortex (2). However, measurements of vascular responses are limited (4). Therefore, we examined whether cortical parenchymal arteries dilate during NBM stimulation using two-photon microscopy. Imaging of cortical vasculature was performed in adult male mice (22 – 38 g, n=7) anesthetized with urethane (1.1 g/kg, i.p.) and artificially ventilated (3). The diameter of single penetrating arteries at different depths (~750 μm, layers I-V) of the frontal cortex was measured (5) and examined changes in the diameter during focal electrical stimulation of the NBM (0.5 ms at 30-50 μA and 50 Hz) (3) and hypercapnia (3% CO2 inhalation). Values are means ± S.E.M., compared by ANOVA. At the resting condition, the diameters of 8 penetrating arteries measured ranged 10 – 28 μm over the cortical depths. The artery showed only a marginal increase in diameter at the cortical surface following stimulation of the NBM in accordance with the previous report (2). In contrast, the artery at a depth of 50 μm from the surface showed an obvious increase in diameter during stimulation of the NBM: the diameter of 15.6 ± 1.6 μm before stimulation increased to 17.6 ± 1.7 μm during NBM stimulation. A significant increase (p<0.05) in arterial diameter by 9 – 13% was observed throughout the different layers of the cortex, except at upper part of layer V, where the diameter of penetrating arteries increased only slightly during NBM stimulation. Hypercapnia caused obvious dilation of the penetrating arteries in all cortical layers, including the surface arteries. The diameters began to increase within 1 sec after the onset of NBM stimulation in the upper cortical layer, and later in lower layers. Our results indicate that activation of the NBM dilates cortical penetrating arteries in a layer specific manner in magnitude and latency, presumably related to the density of cholinergic nerve terminals from the NBM.