The sympathetic neurotransmitter norepinephrine (NE) influences renal sodium excretion via activation of adrenergic receptors. The thick ascending limb (THAL) possesses both -2 and -adrenergic receptors. However, the role(s) different adrenergic receptors play in how isolated THALs respond to NE are unclear. We tested the hypothesis that both -2 and -adrenergic receptors are responsive to NE in the isolated THAL, with -2 receptors inhibiting and -receptors stimulating chloride flux (J_Cl). THALs from male Sprague-Dawley rats were perfused in vitro, and the effects of 1) incremental NE, 2) the -2 agonist clonidine, and 3) the -agonist isoproterenol on J_Cl were measured. Low concentrations (0.1 nM) of NE decreased J_Cl from a rate of 114.2 ± 8.1 to 93.5 ± 14.6 pmol · mm¹ · min¹ (P < 0.05), with the nadir occurring at 1 nM (67.7 ± 8.8 pmol · mm¹ · min¹; P < 0.05). In contrast, greater concentrations of NE significantly increased J_Cl from the nadir to a maximal rate of 131.0 ± 28.5 pmol · mm¹ · min¹ at 10 µM (P < 0.05). To evaluate the adrenergic receptors mediating these responses, the THAL J_Cl response to NE was measured in the presence of selective antagonists of - and -2 receptors. A concentration of NE (1 µM), which alone tended to increase J_Cl, decreased THAL J_Cl (from 148.9 ± 16.4 to 76.2 ± 13.6 pmol · mm¹ · min¹; P < 0.01) in the presence of the -antagonist propranolol. In contrast, a concentration of NE (0.1 µM), which alone tended to decrease J_Cl, increased THAL J_Cl (from 85.5 ± 20.1 to 111.8 ± 20.1 pmol · mm¹ · min¹; P < 0.05) in the presence of the -2 antagonist rauwolscine. To further clarify the role of different adrenergic receptors, selective adrenergic agonists were used. The -2 agonist clonidine decreased J_Cl from 102.4 ± 9.9 to 54.0 ± 15.7 pmol · mm¹ · min¹, a reduction of 49.1 ± 11.0% (P < 0.02). In contrast, the -agonist isoproterenol stimulated J_Cl from 95.3 ± 11.6 to 144.1 ± 15.0 pmol · mm¹ · min¹, an increase of 56 ± 14% (P < 0.01). We conclude that 1) the sympathetic neurotransmitter NE exerts concentration-dependent effects on J_Cl in the isolated rat THAL, 2) selective -2 receptor activation inhibits THAL J_Cl, and 3) selective -receptor activation stimulates THAL J_Cl. These data indicate the response elicited by the isolated rat THAL to NE is dependent on the neurotransmitter concentration, such that application of NE in vitro biphasically modulates J_Cl via differential activation of -2 and -adrenergic receptors in a concentration-dependent manner.