Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca 2+] n), perinuclear ([Ca 2+] pn), and cytoplasmic free calcium ([Ca 2+] i) levels are regulated by the concentration of extracellular free magnesium ions ([Mg 2+] o). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca 2+ responses to stepwise reduction in [Mg 2+] o (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg 2+] o-containing incubation media, basal mean [Ca 2+] i was 89.6±15 nM. Lowering [Mg 2+] o to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca 2+] i going to 213±43, 368±67, 471±77, and 642±98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg 2+] o, the higher the [Ca 2+] i. Restoration of [Mg 2+] o to normal caused decreases in [Ca 2+] i to 215.9±42.3 nM, but only complete removal of [Ca 2+] o returned [Ca 2+] i to basal levels. Results show that basal [Ca 2+] pn (282±92 nM) exceeds basal cytoplasmic Ca 2+ (61±27.8 nM) and [Ca 2+] n (20±7.6 nM). However, reduction of normal [Mg 2+] o to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca 2+] pn (1503±102 nM)>cytoplasmic Ca 2+ (688±49 nM)≡[Ca 2+] n (674±12 nM). Nuclear Ca 2+ rose dramatically (e.g., 35–40 times basal levels). Both verapamil (1 μM) and Ni 2+ (5 mM) prevented, completely, the rises in Ca 2+ in all compartments, suggesting that Mg 2+-dependent Ca 2+ accumulation may be dependent on nuclear, endoplasmic reticulum–Golgi, and cytoplasmic L-type voltage membrane-regulated Ca 2+ channels. The normally low [Ca 2+] n suggests that Ca 2+ does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral–central nervous system pathobiology, and, particularly, alcohol-induced strokes.