In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF-7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg2+ (from 0.5 to 0 mM) and the growth rates were determined by [3H]-thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg2+ depletion whereas the MCF-7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg2+ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF-7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF-7 cells. We also demonstrated that Mg2+ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg2+-restricted HC11 cells are still responsive to the epidermal growth factor (EGF)- and insulin-mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg2+ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27Kip1.