A new fluorescence method has been developed to measure simultaneously and independently the release of fluorophores from two vesicle populations. Calcein and sulforhodamine B were used as a probe couple: the leakage of these probes from vesicles can be recorded independently since they can be excited simultaneously at 510 nm, and their individual fluorescence can be isolated by measuring the fluorescence signal at 525 and 590 nm, using a T-shape fluorometer. Controls show that both probes are suitable for the leakage assay based on fluorescence self-quenching, that they do not interact physically or chemically at the concentrations used in the method, and that they leak in a similar fashion from a given vesicle type. This dual-probe technique is applied to examine the specificity of the release relative to the cholesterol content of the vesicles for melittin, a toxin. This new approach shows in a straightforward manner that melittin-induced release for a given population can be modulated by the presence of vesicles with another lipid composition and this competitive release is associated with a preferential distribution of the peptide on the targeted vesicles.