The mechanisms of Na+ transport across cell membranes were investigated in the in vitro microperfused hamster ascending thin limb (ATL) of Henle's loop using a fluorescent Na+ indicator sodium-binding benzofuran isophthalate. The intracellular Na+ concentration ([Na+]i) of the ATL cells was 17.1 +/- 1.7 mM (n = 22) when the ATL was microperfused in vitro with Hepes-buffered solution containing 204 mM Na+. Elimination of metabolites such as glucose and alanine from the basolateral solution increased [Na+]i. Applying either 5 mM cyanide or 5 mM iodoacetic acid to the bath also increased [Na+]i. The elimination of K+ and the addition of 10(-4) M ouabain in the bath increased [Na+]i by 25.0 +/- 5.0 mM (n = 5) in 3 min and by 10.7 +/- 2.4 mM (n = 4), respectively. The elimination of luminal and basolateral Na+ resulted in a decrease in [Na+]i, indicating Na+ permeability of both the luminal and basolateral cell membranes. The luminal Na+ permeability was not affected by furosemide. The presence of luminal Na+ permeability and the basolateral Na+/K+ ATPase suggests the presence of net active reabsorption of Na+, which is not a physiologically important amount, in our estimation.