Resting Ca²⁺ absorption by cortical thick ascending limbs (CALs) is passive and proceeds through the paracellular pathway. In contrast, parathyroid hormone (PTH) stimulates active, transcellular Ca²⁺ absorption (J _Ca). The Ca²⁺-sensing receptor (CaSR) is expressed on serosal membranes of CALs. In the present study, we tested the hypothesis that activation of the CAL CaSR indirectly inhibits passive Ca²⁺ transport and directly suppresses PTH-induced cellularJ _Ca. To test this theory, we measuredJ _Ca and Na absorption (J _Na) by single perfused mouse CALs. Net absorption was measured microfluorimetrically in samples collected from tubules perfused and bathed in symmetrical HEPES-buffered solutions or those in which luminal Na⁺ was reduced from 150 to 50 mM. We first confirmed that Gd³⁺ activated the CaSR by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) in CALs loaded with fura 2. On stepwise addition of Gd³⁺ to the bath, [Ca²⁺]_i increased, with a half-maximal rise at 30 μM Gd³⁺. J _Ca and transepithelial voltage (V _e,) were measured in symmetrical Na⁺-containing solutions. PTH increasedJ _Ca by 100%, and 30 μM Gd³⁺inhibited this effect. V _e was unchanged by either PTH or Gd³⁺. Similarly, NPS R-467, an organic CaSR agonist, inhibited PTH-stimulated J _Ca without altering V _e. Neither PTH nor Gd³⁺affected J _Na. Addition of bumetanide to the luminal perfusate abolished J _Na andV _e. These results show that CaSR activation directly inhibited PTH-induced transcellular J _Caand that cellular Ca²⁺ and Na⁺ transport can be dissociated. To test the effect of CaSR activation on passive paracellular Ca²⁺ transport, J _Ca was measured under asymmetrical Na conditions, in which passive Ca²⁺ transport dominates transepithelial absorption. PTH stimulated J _Ca by 24% and was suppressed by Gd³⁺. In this setting, Gd³⁺ reducedV _e by 32%, indicating that CaSR activation inhibited both transcellular and paracellular Ca²⁺transport. We conclude that the CaSR regulates both active transcellular and passive paracellular Ca²⁺ reabsorption but has no effect on J _Na by CALs.