Previous micropuncture studies suggested that macula densa (MD) cells might detect variations in luminal sodium chloride concentration ([NaCl]_l) through changes in cytosolic calcium ([Ca²⁺]_c). To test this hypothesis, MD [Ca²⁺]_cwas measured with fluorescence microscopy using fura 2 in the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney. Tubules were bathed and perfused with a Ringer solution, [NaCl]_l was varied and isosmotically replaced withN-methyl-d-glucamine cyclamate. Control [Ca²⁺]_c, during perfusion with 25 mM NaCl and 150 mM NaCl in the bath, averaged 101.6 ± 8.2 nM (n = 21). Increasing [NaCl]_l to 150 mM elevated [Ca²⁺]_cby 39.1 ± 5.2 nM (n = 21,P < 0.01). This effect was concentration dependent between zero and 60 mM [NaCl]_l. The presence of either luminal furosemide or basolateral nifedipine or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a potent Cl⁻ channel blocker, significantly reduced resting [Ca²⁺]_cand abolished the increase in [Ca²⁺]_cin response to increased [NaCl]_l. Nifedipine failed to produce a similar inhibitory effect when added exclusively to the luminal perfusate. Also, 100 nM BAY K 8644, a voltage-gated Ca²⁺ channel agonist, added to the bathing solution increased [Ca²⁺]_cby 33.2 ± 8.1 nM (n = 5,P < 0.05). These observations suggest that MD cells may detect variations in [NaCl]_l through a signaling pathway that includes Na⁺-2Cl⁻-K⁺cotransport, basolateral membrane depolarization via Cl⁻ channels, and Ca²⁺ entry through voltage-gated Ca²⁺ channels.