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+](c) was 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 with N-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+](c) by 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+](c) and abolished the increase in [Ca2+](c) in 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+](c) by 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.