The present studies examined some of the properties of Cl- channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having a PCl/PK ratio of 10 and a unit conductance of 93 pS in symmetric 320 mM KCl. In asymmetric KCl solutions, the I-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl- conductance of isolated medullary thick ascending limb segments. The Cl- channels incorporated into the planar bilayers were asymmetrical: the trans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10 mM, while the cis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50 mM.