The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of lo-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Φo, as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Φo was related to the assumed partition coefficient kp of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with kp = 0 (probe completely excluded from liquid-ordered phase), Φo = 0.16 and with kp = 0.2, Φo = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.