First published September 21, 2001; 10.1152/ajprenal.00357.2001.The macula densa expresses a luminal Na⁺-K⁺-2Cl cotransporter and a basolateral Cl conductance. Although it is known that cotransport of Na⁺, K⁺, and Cl is the first step in tubuloglomerular feedback (TGF), subsequent steps are unclear. We hypothesized that Na⁺-K⁺-2Cl entry via the luminal Na⁺-K⁺-2Cl cotransporter elevates intracellular Cl, increases electrogenic Cl efflux across the basolateral membrane, and depolarizes the macula densa, initiating TGF. We perfused afferent arterioles with macula densa attached. The macula densa was perfused with solutions containing either 5 mM Na⁺ and 3 mM Cl (low NaCl) or 80 mM Na⁺ and 77 mM Cl (high NaCl). When the macula densa perfusate was changed from low to high NaCl, afferent arteriole diameter decreased from 15.8 ± 0.8 to 13.1 ± 0.7 mm (P < 0.05). Adding 10 µM furosemide to the macula densa lumen blocked TGF. When nystatin, a group I cation ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl, it induced TGF (from 18.0 ± 1.5 to 15.6 ± 1.6 mm; P = 0.003). When valinomycin, a K⁺-selective ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl containing 5 mM K⁺, it did not induce TGF. Subsequent addition of 50 mM KCl to the macula densa perfusate induced TGF (from 21.7 ± 0.8 to 17.5 ± 1.3 mm; P = 0.0047; n = 6). Adding 50 mM KCl without valinomycin did not induce TGF. When 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1 µM), a Cl channel blocker, was added to the bath, it blocked TGF induced by high NaCl, but did not block TGF induced by valinomycin plus 50 mM KCl. NPPB did not alter afferent arteriole constriction induced by norepinephrine. We concluded that increased NaCl in the lumen of the macula densa leads to influx of Cl via the Na⁺-K⁺-2Cl cotransporter. The accelerated transport increases intracellular Cl. The subsequent exit of Cl across the basolateral membrane via Cl channels in turn leads to depolarization of the macula densa and thereby induces TGF.