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The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H₁Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca ²⁺ signals drive histamine‐induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca ²⁺ signaling in histamine‐induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca ²⁺‐ and NO‐sensitive dyes, Fura‐2/AM and DAF‐FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca ²⁺ concentration in hCMEC/D3 cells throughout a …