Macula densa (MD) cells detect changes in distal tubular sodium chloride concentration ([NaCl]L), at least in part, through an apical Na:2Cl: K co-transporter. This co-transporter may be a site for regulation of tubuloglomerular feedback (TGF), and recently angiotensin II (Ang II) was shown to regulate the MD Na:2Cl:K co-transporter. In addition, nitric oxide (NO) produced via neuronal NO synthase (nNOS) in MD cells attenuates MD-TGF signaling. This study investigated [NaCl]L-dependent MD-NO production, the regulation of co-transporter activity by NO, and the possible interaction of NO with Ang II. MD cell Na+ concentration ([Na +]i) and NO production were measured using sodium-binding benzofuran isophthalate and 4-amino-5-methylamino-2′,7′ -difluorescein diacetate, respectively, using fluorescence microscopy. Na: 2Cl: K co-transport activity was assessed as the initial rate of increase in [Na +]i when [NaCl]L. was elevated from 25 to 150 mM. 10-4 M 7-nitroindazole, a specific nNOS blocker, significantly increased by twofold the initial rate of rise in [Na+]i when [NaCl]L was increased from 25 to 150 mM, indicating co-transporter stimulation. There was no evidence for an interaction between the stimulatory effect of Ang II and the inhibitory effect of NO on co-transport activity, and, furthermore, Ang II failed to alter MD-NO production. NO production was sensitive to [NaCl]L but increased only when [NaCl]L was elevated from 60 to 150 mM. These studies indicate that MD-NO directly inhibits Na:2Cl:K co-transport and that NO and Ang II independently alter co-transporter activity. In addition, generation of MD-NO seems to occur only at markedly elevated [NaCl]L, suggesting that NO may serve as a buffer against high rates of MD cell transport and excessive TGF-mediated vasoconstriction.