The fluorescent sterol Δ5,7,9,(11)-cholestatrien-3β-ol (cholestatrienol) was incorporated into 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. Previously another fluorescent sterol, dehydroergosterol (F. Schroeder, Y. Barenholz, E. Gratton and T.E. Thompson, Biochemistry 26 (1987) 2441), was used for this purpose. However, there is some concern that dehydroergosterol may not be the best analogue for cholesterol. Fluorescence properties of cholestatrienol in POPC SUV were highly sensitive to cholestatrienol purity. The fluorescence decay of cholestatrienol in the POPC SUV was analyzed by assuming either that the decay is comprised of a discrete sum of exponential components or that the decay is made up of one or more component's distribution of lifetimes. The decay for cholestatrienol in POPC SUV analyzed using distributions had a lower X2 value and was described by a two-component Lorentzian function with centers near 0.86 and 3.24 ns, and fractional intensities of0.96 and 0.04, respec distributions are generated by separate continua of environments for the cholestatrienol molecule described by different dielectric constants. In the range 0-6 mol% cholestatrienol, the cholestatrienol underwent a concentration-dependent relaxation. This process was characterized by red-shifted absorption and maxima and altered ratios of absorption and fluorescence excitation maxima. Fluorescence quantum yield, lifetime, steady-state anisotropy, limiting anisotropy and rotational rate remained constant. In contrast, in POPC vesicles containing between 6 and 33 mol% cholestatrienol, the fluorescent cholestatrienol partially segregated, resulting in quenching. Thus, below 6 mol% cholestatrienol, the cholestatrienol appeared to behave in part as monomers exposed to some degree to the aqueous solvent in a sterol-poor domain within POPC bilayers. Since the lifetim in the sterol-poor domain. The fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy of cholestatrienol in the sterol-poor domain decreased to limiting, nonzero values while the rotational rate increased to a limiting value. Thus, the sterol-poor domain became more disordered when it coexisted with the sterol-rich domain. In POPC vesicles containing 3 mol% cholestatrienol plus increasing mol% cholesterol the two sterols codistributed, since fluorescence quenching was not observed. Above 6 mol% total sterol the cholestatrienol was sensitive to sterol motional properties in the laterally segregated sterol-rich domain. The sterol-rich domain became more rigid with increasing cholesterol content of the POPC SUV. The data are consistent with the presence of a relatively ordered sterol-rich domain and a relatively sterol-poor polarity-sensitive domain coexisting in fluid-phase phospholipid vesicles. In conclusion, cholestatrienol and dehydroergosterol both appear to be sensitive probe molecule for monitoring cholesterol dynamics in membranes. © 1988.