Chloride-dependent cation transport systems in a number of cells and tissues are inhibited by 5-sulfamoylbenzoic acid loop diuretics, such as furosemide and bumetanide. Interactions between chloride and bumetanide have been examined in the catecholamine-activated (Na + K + 2Cl) co-transport pathway of the duck red blood cell. Levels of chloride were varied while maintaining a constant ratio of internal to external chloride across the cell membrane. Increasing external chloride from 20 to 100 mM shifted the dose-response curve for the effect of bumetanide on co-transport toward higher concentrations of the drug. The bumetanide concentration producing half-maximal inhibition (IC50) was increased from approximately 6 X 10(-8) to approximately 2 X 10(-7) M. When cells were incubated in the presence of a constant, submaximal inhibitory dose of bumetanide (10(-8) M), increasing external chloride (in increments of 20 mM) from 20 to 140 mM progressively decreased the level of inhibition of the co-transport system. Kinetic analysis of the data demonstrates that bumetanide and chloride compete for a common site.