ANG II is a modulator of tubuloglomerular feedback (TGF); however, the site of its action remains unknown. Macula densa (MD) cells sense changes in luminal NaCl concentration ([NaCl]_L) via a Na-2Cl-K cotransporter, and these cells do possess ANG II receptors. We tested whether ANG II regulates Na-2Cl-K cotransport in MD cells. MD cell Na⁺ concentration ([Na⁺]_i) was measured using sodium-binding benzofuran isophthalate with fluorescence microscopy. Resting [Na⁺]_i in MD cells was 27.7 ± 1.05 mM (n = 138) and increased ([Na⁺]_i) by 18.5 ± 1.14 mM (n = 17) at an initial rate ([Na⁺]_i/t) of 5.54 ± 0.53 × 10⁴ U/s with an increase in [NaCl]_L from 25 to 150 mM. Both [Na⁺]_i and [Na⁺]_i/t were inhibited by 80% with 100 µM luminal furosemide. ANG II (10⁹ or 10¹² M) added to the lumen increased MD resting [Na⁺]_i and [NaCl]_L-dependent [Na⁺]_i and caused a twofold increase in [Na⁺]_i/t. Bath (10⁹ M) ANG II also stimulated cotransport activity, and there was no additive effect of simultaneous addition of ANG II to bath and lumen. The effects of luminal ANG II were furosemide sensitive and abolished by the AT₁ receptor blocker candesartan. ANG II at 10⁶ M failed to stimulate the cotransporter, whereas increased cotransport activity could be restored by blocking AT₂ receptors with PD-123, 319. Thus ANG II may modulate TGF responses via alterations in MD Na-2Cl-K cotransport activity.