Objective: It is hypothesized that an increase in basolateral Ca²⁺ activates the Ca²⁺-sensing receptor (CaSR) of the thick ascending limb (TAL), leading to a reduction of NaCl reabsorption and a decrease in the lumen-positive transepithelial driving force for paracellular Ca²⁺ and Mg²⁺ reabsorption. In the present study, we examined the effects of Ca²⁺/CaSR activation on mouse TAL. Methods: Experiments were performed in isolated perfused TAL in active or passive transport mode. Results: Under control conditions we did not observe any transport inhibition by Ca²⁺. In the absence of active NaCl transport (luminal perfusion with furosemide), a lumen-to-bath NaCl gradient of 145 versus 30mM induced a lumen-negative diffusion potential (PDte), which was significantly changed when the basolateral Ca²⁺ was elevated from 1 to 5mM (-21±1.2mV vs. -15±1.2mV; n=16). The opposite gradient induced a lumen-positive PDte, which was only slightly reduced when basolateral Ca²⁺ was elevated (17.9±1.4mV vs. 17.5±1.4mV; n= 8). These findings would be compatible with a reduction of paracellular cation selectivity by Ca²⁺. To investigate the involvement of CaSR, the agonist calindol (1mM) and the antagonist Calhex231 (10mM) were tested. Calindol did not change Vte or PDte, and Calhex231 did not block the Ca²⁺-induced change in PDte. Forskolin (1mM)/vasopressin (250mU/ml) or U73122 (1mM) were used to further investigate the CaSR-activated intracellular signalling. Neither stimulation of cAMP formation nor inhibition of PLC influenced the effect of Ca²⁺ on PDte. Conclusion: Even if we consider the contribution of the Ca²⁺ gradient to the diffusion voltage, our data suggest that basolateral Ca²⁺ interferes with the paracellular pathway by reduction of tight junction permselectivity. This effect depends on the direction of the transepithelial voltage/diffusion gradient for Na⁺.