Chlorhexidine did not inhibit ATPase in intact cells of Escherichia coli K12 W1317i-, even at bactericidal concentrations, and ATP hydrolysis was greatest at the highest concentration (40 mg/l), even though no net uptake of substrate occurred. Like dinitrophenol and tribrominated salicylanilide, polymyxin and chlorhexidine collapsed the membrane potential at inhibitory concentrations. Membrane disruption, and not ATPase inactivation, is considered the lethal event in chlorhexidine action.