Cholesterol plays a number of roles in cell membranes, and dehydroergosterol (DHE) is a fluorescent derivative of cholesterol, which is used to investigate the association structure of cholesterol. Although the fluorescent property of DHE depends on its association state, it is insufficient to distinguish the association state of DHE only by its fluorescence. Circular dichroism (CD) spectroscopy is an effective way to investigate the molecular geometry of DHE. In the present study, therefore we investigated the association structure of DHE by CD and fluorescence spectroscopy in solution and in a lipid membrane. DHE was shown to exist as three states (monomeric, microcrystalline, and micelle states) in methanol-water mixtures depending on solvent hydrophobicity. The CD spectrum of DHE in a liposome was similar to that of the micelle state, indicating that the association state of DHE in a liposome exhibits a parallel arrangement similar to that in the micelle state. This result is difficult to be obtained only from the measurement of the fluorescence spectra. The combination of CD and fluorescence spectroscopic techniques is necessary to investigate the association of DHE.