We evaluated the effects of varying aqueous Cl- concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl- channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for channel activity, and the effects of varying Cl- concentrations and/or PGO on the relation between holding voltage (VH, mV) and open-time probability (Po). Reducing cis Cl- concentrations, in the range 50-320 mM, produced a linear reduction in fractional open time (Po) with a half-maximal reduction in Po at cis Cl- approximately 170 mM. Channel activity was sustained by equimolar replacement of cis Cl- with F-, but not with impermeant isethionate. For trans solutions, the relation between Cl- concentration and Po was negatively cooperative, with 50% reduction in po at 10 mM Cl-. Reducing cis Cl- had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent energy (delta G) for channel opening. Phenylglyoxal (PGO) reduced Z and altered delta G for Cl- channel activity when added to cis, but not trans solutions. Furthermore, in the presence of cis PGO, reducing the cis Cl- concentration had no effect on Z but altered delta G. Thus we propose that cis PGO and cis Cl- concentrations affect separate sites determining channel activity at the extracellular faces of these Cl- channels.