By the use of frequency domain cross-correlation fluorometry, the fluorescence lifetime of the water soluble probe 8,1-anilinonapthalene sulfonic acid (ANS) in aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and phosphatidylethanolamine transphosphatidylated from egg phosphatidylcholine (TPE) was measured. The orientational order parameter and rotational diffusion constant of the lipophilic probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were also determined in TPE dispersions. In agreement with a previous study on DOPE (Cheng (1989) Biophys. J. 55, 1025–1031), abrupt changes in both the order packing and rotational diffusion constant were found at the lamellar liquid crystalline (Lα) to inverted hexagonal (HII) phase transition of TPE. Owing to the subnanosecond resolution capability of this frequency domain fluorometric technique, the heterogeneous fluorescence decay of ANS was resolved into three distinct components with different decay lifetimes (τ's). They were 0 < τ < 0.5 ns, 2 < τ < 9 ns and τ > 15 ns. These lifetime regions were attributed to the partitioning of ANS into the bulk aqueous medium, the lipid/water interface and the lipid hydrocarbon region, respectively. These classifications of lifetime regions were further supported by the sensitivity of those lifetime components with the solvent isotopic shift of D2O. Similar to the changes of orientational order and rotational diffusion of lipophilic probe, the lifetime and intensity fraction of ANS associated with the lipid/water interfacial region declined abruptly at the Lα-HII transition of both DOPE and TPE. This observation suggested that a dehydration of the lipid headgroup surface occurs at the Lα-H11 transition. This study provided evidence that both the lipid headgroup surface hydration and the lipid dynamics change drastically as a result of the macroscopic rearrangement of lipids at the Lα-HII transition.