The kinetics of carboxyfluorescein efflux induced by the amphipathic peptide δ-lysin from vesicles of porcine brain sphingomyelin (BSM), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol (Chol) were investigated as a function of temperature and composition. Sphingomyelin (SM)/Chol mixtures form a liquid-ordered (Lo) phase whereas POPC exists in the liquid-disordered (Ld) phase at ambient temperature. δ-Lysin binds strongly to Ld and poorly to Lo phase. In BSM/Chol/POPC vesicles the rate of carboxyfluorescein efflux induced by δ-lysin increases as the POPC content decreases. This is explained by the increase of δ-lysin concentration in Ld domains, which enhances membrane perturbation by the peptide. Phase separations in the micrometer scale have been observed by fluorescence microscopy in SM/Chol/POPC mixtures for some SM, though not for BSM. Thus, δ-lysin must detect heterogeneities (domains) in BSM/Chol/POPC on a much smaller scale. Advantage was taken of the inverse variation of the efflux rate with the Ld content of BSM/Chol/POPC vesicles to estimate the Ld fraction in those mixtures. These results were combined with differential scanning calorimetry to obtain the BSM/Chol/POPC phase diagram as a function of temperature.