The mechanisms responsible for transepithelial Ca<sup>2+</sup> and Mg<sup>2+</sup> transport in the isolated perfused cortical thick ascending limb (cTAL) of Henle’s loop of the mouse nephron were investigated by measuring transepithelial voltages (PD<sub>te</sub>) and transepithelial ion net fluxes (J<sub>Na</sub>, J<sub>c1</sub>, J<sub>ĸ</sub>, J<sub>ca</sub>, J<sub>Mg</sub>) by electron microprobe analysis. In the presence of furosemide (10<sup>-4</sup> mol·l<sup>-1</sup>, lumen) and diphenylamine-2-carboxylate (DPC, 10<sup>-4</sup> mol·l<sup>-1</sup>, bath), known inhibitors of NaCl reabsorption in the TAL, Ca<sup>2+</sup> and Mg<sup>2+</sup> reabsorption was completely inhibited. In the presence of furosemide, J<sub>Ca</sub> fell from 0.75 ± 0.07 to -0.08 ± 0.09 pmol·min<sup>-1</sup>.mm<sup>-1</sup> (n = 5) and J<sub>Mg</sub> from 0.47 ± 0.04 to -0.01 ± 0.11 pmol·min<sup>-1</sup>.mm<sup>-1</sup> (n = 5). In the presence of DPC, J<sub>Ca</sub> fell from 0.57 ± 0.08 to -0.07 ± 0.11 pmol·min<sup>-1</sup>.mm<sup>-1</sup> (n = 5), and J<sub>Mg</sub> from 0.16 ± 0.02 to-0.11 ± 0.07pmol·min<sup>-1</sup>·mm<sup>-1</sup>(n = 5). With furosemide, inhibition of Ca<sup>2+</sup> and Mg<sup>2+</sup> transport was paralleled by a 93% inhibition of NaCl reabsorption, while in the presence of DPC there was a 60% reduction of NaCl reabsorption. These effects were fully reversed after removal of the inhibitors from the lumen or bath solutions. In the absence of active NaCl transport, a lumen-to-bath directed-NaCl gradient (lumen: 150 m M NaCl + furosemide, bath: 50 m M NaCl + 200 m M mannitol) generated a negative transepithelial dilution potential of-13.8 ± 1.1 mV (n = 8) which induced a significant Ca<sup>2+</sup> and Mg<sup>2+</sup> secretion into the tubular lumen of -0.59 ± 0.06 and 0.43 ± 0.05pmol·min<sup>-1</sup>·mm<sup>-1</sup> (n = 8), respectively. Abath-to-lumen-directed NaCl gradient on the other hand (lumen: 50 m M NaCl + furosemide, bath: 150m M NaCl) generated a positive transepithelial dilution potential of +15.9 ± 0.6 mV (n = 7), inducing a significant Ca<sup>2+</sup> and Mg<sup>2+</sup> reabsorption of 0.62 ± 0.08 and 0.38 ± 0.07 pmol·min<sup>-1</sup> mm<sup>-1</sup>(n = 7), respectively. Linear regression analysis of individual Ca<sup>2+</sup> and Mg<sup>2+</sup> net flux data versus voltage indicated that J<sub>Ca</sub> and J<sub>Mg</sub> were highly correlated to PD<sub>te</sub>. In conclusion, these data indicate that transepithelial Ca<sup>2+</sup> and Mg<sup>2+</sup> reabsorption in the mouse cTAL is predominantly a passive process, driven by the lumen-positive PD<sub>te</sub>.