Basal free Mg2+ concentration was 0.49 +/- 0.03 mM in normal single Madin-Darby canine kidney (MDCK) cells as measured by fluorescence with the aid of mag-fura-2. Accordingly, Mg2+ may enter the cell down a transmembrane electrical gradient. The present study describes some aspects of Mg2+ entry into the established MDCK cell line. MDCK cells were Mg2(+)-depleted (0.26 +/- 0.01 mM) by culturing in Mg2(+)-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCl2, and intracellular Mg2+ concentration ([Mg2+]i) was monitored with fluorescence. [Mg2+]i returned to normal basal levels, 0.56 +/- 0.05 mM, with a refill rate of 272 +/- 39 nM/s, n = 4. Mg2+ entry was not changed by 5.0 mM external Ca2+ but was completely inhibited with 5.0 mM La3+. Intracellular Ca2+ concentration was not altered by Mg2+ depletion or during Mg2+ repletion. Mg2+ uptake was inhibited by verapamil (0 +/- 27 nM/s, n = 3), was inhibited less so by diltiazem (141 +/- 34 nM/s, n = 3), and was not affected by nifedipine (300 +/- 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg2+]i returned to normal levels. These data indicate the presence of a unique Mg2+ entry pathway in MDCK cells that may be important in Mg2+ homeostasis. The model of Mg2+ refill into Mg2(+)-depleted cells may be useful in other cell types.