Next, we studied the hydrocarbon chain region with diphenylhexatriene (DPH), which is a rod-like hydrophobic fluorescent probe with simple geometry and directional emission of polarized light. It orients along the lipid hydrocarbon chains and measures their ordering and dynamics-both indicative of the membrane phase. Our previous studies using timeresolved anisotropy measurements demonstrated great sensitivity of DPH to the ordering effect of different amounts and different species of sterols in lipid bilayer. We measured the temperature-dependence of steady-state anisotropy emission spectra of DPH embedded in unilamellar vesicles. The anisotropy values obtained by averaging the spectra (Fig. ) are shown in Fig. . They show a gradual decrease in anisotropy upon temperature increase, reflecting a decreased order and/or increased dynamics of the lipid aliphatic tails. Noteworthy, the qualitative similarity of the results obtained using Patman generalized polarization and DPH anisotropy reassures that the observed changes can be detected both in the bilayer core, but also closer to the headgroup region, and are not limited to a single fluorescent probe or detection method used. The data can again be fitted by the modified Boltzmann sigmoidal functions, whose curvatures change sign at similar temperatures as those fitted to the GP data (Fig. ). Curiously, Patman reports this to take place at a slightly lower temperature, indicating that the hexagonal packing of DPPC chains initiates from the carbonyl region probed by Patman, before it proceeds to the chain region proved by DPH. All the fitting parameters are provided in Table .