Resting Ca(2+) absorption by cortical thick ascending limbs (CALs) is passive and proceeds through the paracellular pathway. In contrast, parathyroid hormone (PTH) stimulates active, transcellular Ca(2+) absorption (J(Ca)). The Ca(2+)-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 Ca(2+) transport and directly suppresses PTH-induced cellular J(Ca). To test this theory, we measured J(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 Gd(3+) activated the CaSR by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) in CALs loaded with fura 2. On stepwise addition of Gd(3+) to the bath, [Ca(2+)](i) increased, with a half-maximal rise at 30 microM Gd(3+). J(Ca) and transepithelial voltage (V(e),) were measured in symmetrical Na(+)-containing solutions. PTH increased J(Ca) by 100%, and 30 microM Gd(3+) inhibited this effect. V(e) was unchanged by either PTH or Gd(3+). Similarly, NPS R-467, an organic CaSR agonist, inhibited PTH-stimulated J(Ca) without altering V(e). Neither PTH nor Gd(3+) affected J(Na). Addition of bumetanide to the luminal perfusate abolished J(Na) and V(e). These results show that CaSR activation directly inhibited PTH-induced transcellular J(Ca) and that cellular Ca(2+) and Na(+) transport can be dissociated. To test the effect of CaSR activation on passive paracellular Ca(2+) transport, J(Ca) was measured under asymmetrical Na conditions, in which passive Ca(2+) transport dominates transepithelial absorption. PTH stimulated J(Ca) by 24% and was suppressed by Gd(3+). In this setting, Gd(3+) reduced V(e) by 32%, indicating that CaSR activation inhibited both transcellular and paracellular Ca(2+) transport. We conclude that the CaSR regulates both active transcellular and passive paracellular Ca(2+) reabsorption but has no effect on J(Na) by CALs.