tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg²⁺ balance, we determined the effects of adenosine on cellular Mg²⁺ uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg²⁺ uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg²⁺ uptake, MDCT cells were first Mg²⁺ depleted to 0.22 ± 0.01 mM by being cultured in Mg²⁺-free media for 16 h and then placed in 1.5 mM MgCl₂; next, changes in intracellular Mg²⁺ concentration ([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 137 ± 16 nM/s. Adenosine stimulates basal Mg²⁺ uptake by 41 ± 10%. The selective A₁ purinoceptor agonist N⁶-cyclopentyladenosine (CPA) increased intracellular Ca²⁺ and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg²⁺ uptake. On the other hand, the selective A₂ receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg²⁺ entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg²⁺ uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A₂ agonist-mediated Mg²⁺ entry. Aldosterone potentiated CGS-mediated Mg²⁺ entry, and elevation of extracellular Ca²⁺ diminished CGS-responsive cAMP formation and Mg²⁺ uptake. Accordingly, MDCT cells possess both A₁ and A₂ purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg²⁺ uptake in MDCT cells through separate A₁ and A₂ purinoceptor pathways.