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Abstract The distal convoluted tubule reabsorbs significant amounts of filtered magnesium that is under hormonal control. In this study, we describe the effects of aldosterone on Mg21 uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg21 entry rate into MDCT cells, they were first Mg21 depleted ([Mg21]i, 0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5mMMgCl2. The rate of change in [Mg21]i as measured as a function of time, d([Mg21]i)/dt, was 164 6 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg21 entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg21 uptake (172 6 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg21 uptake rate up to 330 6 39 and 224 6 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg21 entry in MDCT cells, it is inferred that aldosterone may stimulate Mg21 uptake through intracellular signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone potentiation of hormone stimulation of Mg21 uptake and cAMP accumulation. These studies with MDCT cells suggest that aldosterone may modulate the effects of hormones acting within the distal convoluted tubule to control magnesium absorption.