Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (D(w)), the curvature-related hopping diffusion constant (D(H)), and the two local orientational order parameters ( and ) of 1-palmitoyl-2-[[2-[4-(6- phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn- phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of , , and D(H) for DOPE were found to increase significantly at ~12 °C, the known lamellar liquid crystalline (L(α)) to inverted hexagonal (H(II)) phase transition temperature of DOPE. Similar features as well as a decline of D(w) were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 °C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 °C) and DOPE/DOPC (0-100% DOPE at 3 °C), which remained in the L(α) phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.