It has recently been shown that membrane ionic transport pathways of macula densa cells can be measured using conventional microelectrodes. To determine if conductances could be identified at the basolateral membrane of macula densa cells, cortical thick ascending limbs (CTAL) with attached glomeruli were continuously perfused with a 25 mM NaCl bicarbonate-free Ringer solution. Individual basolateral Na+, Cl-, NaCl, and K+ concentrations were altered by isosmotic replacement with N-methyl-D-glucamine and/or cyclamate. Reduction in basolateral [Na+] from 150 to 25 mM hyperpolarized basolateral membrane potential (V(bl)) by 9.9 ± 1.3 mV (n = 10; all data are corrected for changes in liquid junction potential at bath electrode). A decrease in bath [Cl-] from 150 to 25 mM depolarized V(bl) by 20 ± 2.4 mV (n = 13), whereas decreases in bath [NaCl] from 150 to 25 mM depolarized V(bl) by 29 ± 6.8 mV (n = 5). In the presence of 150 mM NaCl bathing solution, a stepwise increase in [K+] from 5 to 15 mM (by replacement of 10 mM NaCl with 10 mM KCl) depolarized V(bl) by 3.3 ± 1.1 mV (n = 8). After correction for individual transepithelial diffusion potentials, Cl- conductance averaged 59 ± 19% of the total basolateral conductance, whereas K+ (23 ± 8%) and Na+ (17 ± 10%) contributed significantly less to the overall basolateral conductance. These results indicate that membrane potential of macula densa cells may be very sensitive to alterations in intracellular Cl- activity and suggest that apical transport of NaCl through a furosemide-sensitive Na+-K+-2Cl- transporter may affect membrane potential in macula densa cells via a change in intracellular Cl- activity.