The activities of several known Ca²⁺ entry and release channels are affected by oxidant stress and therefore may link cellular redox state and Ca²⁺ homeostasis. Redox sensitivity of Ca²⁺ signaling pathways is not only important pathophysiologically, but also during membrane receptor stimulation, when reactive oxygen species (ROS) function as intracellular second messengers. Previous work has shown that a vascular NADPH oxidase plays an important role in agonist-stimulated ROS production. We have studied whether ROS affect agonist- stimulated Ca²⁺ signaling in human aortic endothelial cells (HAEC) stimulated by histamine [Hu Q, et. al. (2002) J Biol Chem 277,32546-32551]. Histamine (1 μM) increased the fluorescence of 2′,7′-dihydrodichlorofluorescin diacetate in HAEC, an indicator of ROS production. This was partially inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 μM), by the farnesyltransferase inhibitor H-Ampamb-Phe-Met-OH (2 μM), and in HAEC transiently expressing Rac1^N17, a dominant negative allele of the protein Rac1, which is essential for NADPH oxidase activity. In indo 1-loaded HAEC, 1 μM histamine triggered oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i) that were blocked by DPI or by H-Ampamb-Phe-Met-OH. Histamine-stimulated ([Ca²⁺]_i) oscillations were not observed in HAEC lacking functional Rac1 protein but were observed when transfected cells were simultaneously exposed to a low concentration of hydrogen peroxide (H₂O₂, 10 μM), which by itself did not alter either [Ca²⁺]_i or levels of inositol 1,4,5-trisphosphate (IP₃). Stimulation of the NADPH oxidase by NADPH stimulated a time- and concentration-dependent increase in superoxide and H₂O₂ production by HAEC that was blocked by DPI and was significantly attenuated in cells transiently expressing Rac1^N17. In permeabilized Mag-indo 1-loaded cells, NADPH and H₂O₂ each decreased the threshold concentration of IP₃ required to release intracellularly stored Ca²⁺ and shifted the IP₃-Ca²⁺ release dose-response curve to the left [Hu Q, et. al. (2002) J Biol Chem 275,15749-15757]. Concentrations of H₂O₂ as low as 3 μM increased the sensitivity of intracellular Ca²⁺ stores to IP₃ and decreased the IP₃ EC₅₀ from 423.2 ± 54.9 to 276.9 ± 14.4 nM (mean ± SD, n = 6, p < 0.05). At a concentration of 100 μM, NADPH decreased the EC₅₀ in response to IP₃ by more than 50% to 170.6 ± 23.2 nM, n = 3, p < 0.05). The effect of NADPH on IP₃-stimulated Ca²⁺ release was blocked by catalase and by DPI and was not observed in cells lacking functional Rac1 protein. These data suggest that histamine generates ROS in HAEC at least partially via NADPH oxidase activation. NADPH oxidase-derived ROS are critical to the generation of [Ca²⁺]_i oscillations in HAEC during histamine stimulation, perhaps by increasing the sensitivity of the endoplasmic reticulum to IP₃. The sensitivity of the IP₃ receptor to oxidative stress may play an important role in cell signaling during membrane receptor stimulation and may be crucial to the downstream activation of Ca²⁺-sensitive transcription factors like NF-κB when the vascular endothelium responds to inflammatory stimuli.