We investigated the dependence of ANG II (10⁻⁸ M)-induced constriction of outer medullary descending vasa recta (OMDVR) on membrane potential (Ψm) and chloride ion. ANG II depolarized OMDVR, as measured by fully loading them with the voltage-sensitive dye bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC₄(3)] or selectively loading their pericytes. ANG II was also observed to depolarize pericytes from a resting value of −55.6 ± 2.6 to −26.2 ± 5.4 mV when measured with gramicidin D-perforated patches. When measured with DiBAC₄(3) in unstimulated vessels, neither changing extracellular Cl⁻ concentration ([Cl⁻]) nor exposure to the chloride channel blocker indanyloxyacetic acid 94 (IAA-94; 30 μM) affected Ψm. In contrast, IAA-94 repolarized OMDVR pretreated with ANG II. Neither IAA-94 (30 μM) nor niflumic acid (30 μM, 1 mM) affected the vasoactivity of unstimulated OMDVR, whereas both dilated ANG II-preconstricted vessels. Reduction of extracellular [Cl⁻] from 150 to 30 meq/l enhanced ANG II-induced constriction. Finally, we identified a Cl⁻channel in OMDVR pericytes that is activated by ANG II or by excision into extracellular buffer. We conclude that constriction of OMDVR by ANG II involves pericyte depolarization due, in part, to increased activity of chloride channels.