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Abstract Basal free Mg2’ concentration was 0.49 t 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 t 0.01mM) by culturing in Mg2’-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCL,, and intracellular M$+ concentration ( [ Mg2+] i) was monitored with fluoresence. [Mg+]i returned to normal basal levels, 0.56 t 0.05 mM, with a refill rate of 272 t 39 nM/s, n = 4. M$+ 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 Mg+ depletion or during M$+ repletion. Mg2+ uptake was inhibited by verapamil (0 t 27 nM/s, n = 3), was inhibited less so by diltiazem (141 t 34 nM/s, n = 3), and was not affected by nifedipine (300 t 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg”‘]i returned to normal levels. These data indicate the presence of a unique M$+ entry pathway in MDCK cells that may be important in M$+ homeostasisT. he model of Mg2+ refill into M$+- depleted cells may be useful in other cell types.