It has been demonstrated that an increase in the extracellular Ca²⁺ concentration reduces reabsorption of Ca²⁺ and Mg²⁺ as well as NaCl via Ca²⁺-sensing receptor (CaSR) in the thick ascending limb (TAL) of kidney. In the present study, we examined the effects of CaSR activation on paracellular permeability in mouse TAL. In isolated perfused TAL from C57Bl6 mice, increasing basolateral Ca²⁺ from 1 to 5mM significantly reduced the permeability ratio P_Na/P_Cl (3.60.4 at 1mM vs 2.50.3 at 5mM; p<0.01), reflected by a decrease in the diffusion potential under asymmetrical NaCl perfusion conditions. The CaSR agonist neomycin induced the similar effect (P_Na/P_Cl: 3.50.6 absence vs 2.50.4 presence; p<0.01). It has been reported that CaSR signalling uses cAMP as well as phospholipase C (PLC) pathways. Stimulation of CaSR results in a decrease in cAMP and/or an increase in IP₃/DAG/cytosolic Ca²⁺ activity. In the presence of 250mU/ml vasopressin and 1mM forskolin, which stimulate cAMP formation, the high Ca²⁺-induced effect was persistent. Pretreatment of the tubules with 1mM U73122, a PLC inhibitor, did not abolish this effect either. Claudin-16 (CLDN16) is a functionally relevant component of TAL tight junctions and confers cation selectivity. In the model of CLDN16 deficiency we observed a significantly attenuated effect of Ca²⁺ on P_Na/P_Cl. Our data suggest that extracellular Ca²⁺ regulates paracellular permeability in mouse TAL involving CLDN16, independent of PLC and cAMP signalling.