A novel method for investigating phase behavior in a ternary lipid membrane was developed and tested. Sixty-five model membranes composed of sphingomyelin (SM), dioleoyl phosphatidylcholine (DOPC) and cholesterol (Ch) were prepared, and fluorescence anisotropy between 25 degrees C and 60 degrees C was measured. Observed fluorescence anisotropy curves as functions of temperature were analyzed using a nonlinear response surface method and Kohonen's self-organizing map. Thus, we generated a scatter plot indicating the distribution of membranes with similar membrane properties. The scatter plot showed that the SM/DOPC/Ch membranes resolved into six clusters with distinct membrane properties. We then conducted differential scanning calorimeter (DSC) measurement of membranes typical of the clusters. The results indicated that the membranes consisted of several phase domains (i.e., L-alpha, L-beta, l(0) phase domains), and the clusters were distinguished by differences in the type and content of membrane domain. This method is accurate because the clusters were determined based on experimental values. This technique is useful for elucidating the phase behavior of ternary lipid membranes. These findings contribute to clarification of domain formation. (C) 2009 Elsevier Inc. All rights reserved.