Regulation of intracellular Ca2+ in breast cancer may be important in modulating cell proliferation, differentiation, apoptosis, and cytotoxicity, as well as contributing to mechanisms of action of anticancer agents. One of these agents, the steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is intimately involved in maintaining cellular Ca2+ homeostasis. The purpose of this study was to investigate Ca2+ regulatory pathways in breast cancer cells and to determine the role of 1,25(OH)2D3 in modulating these pathways. We examined pathways for Ca2+ entry from the extracellular space and Ca2+ mobilization from intracellular stores in the estrogen-receptor negative human breast cancer cell line BT-20. Fluorescence digital video imaging and Ca2+ indicator fura-2 were employed to measure the concentration of intracellular free Ca2+ ([Ca2+]i) and Ca2+ responses at the single-cell level. We found that BT-20 breast cancer cells expressed nonselective, voltage-insensitive Ca2+ channels (VICC), as indicated by their permeability to Mn2+, response to elevated extracellular Ca2+ with an increase in [Ca2+]i, blockage by La3+ and Ni2+, and response to K+ depolarization with a slight decrease in [Ca2+]i and Ca2+ influx. There was no evidence for voltage- dependent Ca2+ channels in BT-20 cells. Endoplasmic reticulum Ca2+ stores comprised a major intracellular Ca2+ pool, as was evident after application of a Ca2+ ionophore ionomycin in nominally Ca2+-free buffer to the cells with thapsigargin-depleted Ca2+ stores. Thapsigargin depletion of Ca2+ stores did not increase influx of extracellular Ca2+, implying no significant activation of the capacitative Ca2+ entry. 1,25(OH)2D3 did not induce a rapid rise in [Ca2+]i, yet Ca2+ influx through VICC was increased. Treatment with 1,25(OH)2D3 for 4-72 h significantly increased the percentage of cells with a markedly elevated basal [Ca2+]i. Ca2+ response of those cells to thapsigargin was attenuated. Taken together, our findings show that VICC and the thapsigargin-sensitive endoplasmic reticulum Ca2+ stores are the principal pathways for Ca2+ entry and Ca2+ mobilization in the breast cancer cell line used in this study. 1,25(OH)2D3 rapidly increases Ca2+ influx through VICC and after a chronic treatment, depletes endoplasmic reticulum Ca2+ stores. Targeting of Ca2+ signaling mediated by VICC and endoplasmic reticulum Ca2+ stores may represent a novel approach to the treatment and chemoprevention of breast cancer.