A fluorescence technique that measures fast ion fluxes across liposome membranes was used to determine the molecularity of the colicin E1 channel. The rate of flux of Tl+ (used as a K+ analogue) into large unilamellar vesicles was measured by its ability to quench the fluorescence of a membrane-impermeable fluorophore entrapped in the vesicles. The dependence of Tl+ flux rate on the concentration of ionophore in the vesicle suspension reveals the molecularity of the ionophore. The method is demonstrated with two ionophores, valinomycin and gramicidin, whose molecularities are known to be one and two, respectively. The molecularity of the colicin E1 channel was determined to be one. This method can be used to study the properties of any ionophore that mediates K+ flux.