Bilayer packing defects in binary dilinoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylcholine (DLPE/POPC) lipid mixtures have been studied by the use of nanosecond-resolved intramolecular excimer fluorescence spectroscopy. Frequency-domain fluorescence intensity decays of dual-chain labelled dipyrenyl lipids of different chain lengths in DLPE/POPC mixtures were acquired at both the monomer (392 nm) and excimer (475 nm) emission channels and at 20°C. On the basis of a new intramolecular excimer formation kinetic model, the extent of aggregation and the rotational mobility, in terms of the equilibrium constant of the monomer to aggregated state and the excimer association rate constant, respectively, of the intralipid pyrenes were calculated from the frequency-domain data. Within the range of 60–100% DLPE where bilayer defects are known to coexist with bilayer and non-bilayer states, a prominent peak in the equilibrium constant and a concomitant dip in the excimer association constant at ∼80% DLPE were observed. Our nanosecond-resolved fluorescence results suggest that the intramolecular excimer kinetic parameters of dipyrenyl lipids are very sensitive to the onset of bilayer packing defects in lipid membranes. Moreover, the onset of bilayer defect state is characterized by the greater extent of aggregation and more hindered rotational mobility of the acyl chains as compared with the bilayer (0% DLPE) and non-bilayer inverted hexagonal (100% DLPE) states of the lipid membranes.