Little attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate -sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.