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Abstract Magnesium reabsorption and regulation within the kidney occur principally within the cortical thick ascending limb (cTAL) cells of the loop of Henle. Fluorometry with the dye, mag-fura- 2, was used to characterize intracellular Mg2″ concentration (IMg2j11) in single cTAL cells. Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat anti-human Tamm-Horsfall and rabbit anti-goat IgG antibodies). Basal [Ig2+"] was 0.52±0.02 mM, which was – 2% of the total cellular Mg. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Mg2j1i, 0.48±0.02, in the normal range. However, cells cultured in nominally magnesium- free media possessed [Mg2J1j, 0.27±0.01 mM, which was associated with a significant increase in net Mg transport, (control, 0.19±0.03 and low Mg, 0.35±0.01 nmol. mg-' protein min-') as assessed by 'Mg uptake. Mg2'-depleted cells were subsequently placed in high Mg solution (5 mM) and the Mg2" refill rate was assessed by fluorescence. [Mg2"1 returned to normal basal levels, 0.53±0.03 mM, with a refill rate of 257±37 nM/s. Mg2" entry was not changed by 5.0 mM Ca2" or 2 mM Sr2+, Cd2+, Co2+, nor Ba2+ but was inhibited by Mn2+ = La3+ .,Gd3+ Ni2+ , Zn2+ Be2+ at 2 mM. Intracellular Ca2` and "Ca uptake was not altered by Mg depletion or Mg2+ refill, indicating that the entry is relatively specific to Mg2e. Mg2+ uptake was inhibited by nifedipine (117±20 nM/s), verapamil (165±34 nM/s), and diltiazem (194±19 nM/s) but enhanced by the dihydropyridine analogue, Bay K 8644 (366±71 nM/s). These antagonists and agonists were reversible with removal and IMg2+Jj subsequently returned to normal basal levels. Mg2+ entry rate was concentration and voltage dependent and maximally stimulated after 4 h in magnesium-free media. Cellular magnesium depletion results in increases in a Mg2+ refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Mg2+ entry pathways in cTAL cells which are specific for Mg2` and highly regulated. These entry pathways are likely involved with renal Mg2` homeostasis. (J. Clin. Invest. 1991. 88:1255-1264.)