-Adrenergic agonists influence electrolyte reabsorption in the proximal tubule, loop of Henle, and distal tubule. Although isoproterenol enhances magnesium absorption in the thick ascending limb, it is unclear what effect, if any, -adrenergic agonists have on tubular magnesium handling. The effects of isoproterenol were studied in immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg²⁺ uptake with fluorescence techniques. Intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured in single MDCT cells by using microfluorescence with mag-fura-2. To assess Mg²⁺ uptake, MDCT cells were first Mg²⁺ depleted to 0.22 ± 0.01 mM by culturing in Mg²⁺-free media for 16 h and then placed in 1.5 mM MgCl₂, and the changes in [Mg²⁺]_i were determined. [Mg²⁺]_i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg²⁺]_i)/dt, of 168 ± 11 nM/s. Isoproterenol stimulated Mg²⁺ entry in a concentration-dependent manner, with a maximal response of 252 ± 11 nM/s, at a concentration of 10⁷ M, that represented a 50 ± 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg²⁺ uptake was completely inhibited with RpcAMPS, a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially blocked by RO 31-822, a protein kinase C inhibitor. Accordingly, isoproterenol-mediated Mg²⁺ entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenol-stimulated Mg²⁺ uptake (326 ± 31 nM/s), whereas elevation of extracellular Ca²⁺ inhibited isoproterenol-mediated cAMP accumulation and Mg²⁺ uptake, 117 ± 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg²⁺ uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.