The article reports the results of the study focusing on age-related changes in the contribution of NO, synthesized by various forms of nitric oxide synthases (NOS) and exogenous L-arginine in acetylcholine-induced dilation of cerebral vessels in rats. A comparative assessment of the responses of pial arteries of various diameters to acetylcholine chloride (ACh, 10–7 M, 8 min) was carried in the absence and in the presence of NOS blockade and exogenous L-arginine (0.25 mM, 30 min) in Sprague-Dawley rats aged 4 and 18 months. NOS blockade was performed by using a non-selective NOS inhibitor (L-NAME, 10–3 M, 12 min) and an inducible NOS inhibitor (aminoguanidine, 1 mM, 10 min). The change in the number and degree of arterial dilation was assessed by measuring the width of the erythrocyte flow in three separate groups of arteries: small (<20 μm in diameter), medium (20–40 μm), and large (>40 μm). It was found that in 18-month-old rats, the contribution of NO, synthesized by constitutive forms of NOS, decreases in the implementation of ACh-mediated endothelium-dependent dilatatory reactions in small and medium pial arteries, while an increase was observed for pial. At the same time, the contribution of inducible NOS to dilation of all types of arteries is on the increase. These processes are accompanied by a decrease in the bioavailability of exogenous L-arginine for NOS.