About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na⁺ (P_Na⁺) and Cl^- (P_Cl^-) permeabilities are unknown. To address this, we measured the effect L-arginine (0.5mmol/L; NO synthase substrate) and cGMP (0.5mmol/L) on P_Na⁺ and P_Cl^- calculated from transepithelial resistance (Rt) and P_Na⁺/P_Cl^-. Rt was 7722±1554 ohm-cm in the control period and 6318±1757 ohm-cm after L-arginine treatment (p<0.05). P_Na⁺/P_Cl^- was 2.0±0.2 in the control period and 1.7±0.1 after L-arginine (p<0.04). Calculated P_Na⁺ and P_Cl^- were 3.52±0.2 x10-5 cm/sec and 1.81±0.10 x10-5 cm/sec respectively in the control period. After L-arginine they were 6.65±0.69 x10-5 cm/sec (p<0.0001 vs control) and 3.97±0.44 x10-5 cm/sec (p<0.0001), respectively. NOS inhibition with L-NAME (5mmol/L) prevented L-arginine's effect on Rt. Next we tested the effect of cGMP. Rt in the control period was 7592±1470 ohm-cm and 4796±847 ohm-cm after dibutyryl-cGMP (0.5mmol/L; db-cGMP) treatment (p<0.04). P_Na⁺/P_Cl^- was 1.8±0.1 in the control period and 1.6±0.1 after db-cGMP (p<0.03). P_Na⁺ and P_Cl^- were 4.58±0.80 x10-5 cm/sec and 2.66±0.57 x10-5 cm/sec, respectively, for the control period, and 9.48±1.63 x10-5 cm/sec (p<0.007) and 6.01±1.05 x10-5 cm/sec (p<0.005), respectively, after db-cGMP. We modeled NO's effect on luminal Na⁺ concentration along the thick ascending limb. We found that NO's effect on the paracellular pathway reduces net Na⁺ reabsorption, and that the magnitude of this effect is equal to that due to NO's inhibition of transcellular transport.