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Abstract Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg21 transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg21-depleted cells. Intracellular free Mg21 concentration ([Mg21]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg21 uptake, MDCT cells were first Mg21 depleted (0.22 6 0.01 mM) by culturing in Mg21-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg21]i was determined. [Mg21]i returned to basal levels, 0.53 6 0.02 mM, with a mean refill rate, d([Mg21]i)/dt, of 164 6 5 nM/s. Both glucagon and AVP stimulated Mg21 uptake into MDCT cells, 196 6 11 and 189 6 6 nM/s, respectively, at concentrations of 3 3 1027 M and 1027 M, respectively. Enhanced Mg21 uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg21 entry was not dependent on protein synthesis. 8-Bromo-cAMP, 1024 M, enhanced Mg21 uptake (225 6 13 nM/s), whereas phorbol testers were without effect. Finally, protein kinaseAinhibition prevented glucagon and AVP stimulation of Mg21 uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg21 uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.