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Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca²⁺‐dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose‐dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was prevented by the genetic blockade of M5 muscarinic receptors (M5‐mAchRs), which was the only mAchR isoform coupled to phospholipase Cβ (PLCβ) present in hCMEC/D3 cells. A comprehensive real‐time polymerase chain reaction analysis revealed the expression of the transcripts …