The formation mechanism of the interdigitated (L beta I) phase and the responses of the individual groups of phospholipids to the phase transition are or basic concern within the community of lipid research. In this work, we studied the effect of acetonitrile (CH3CN) on the structure and phase behavior of dipalmitoyl-phosphatidylcholine (DPPC) bilayers by using differential scanning calorimetry, synchrotron X-ray diffraction, and Fourier transform infrared spectroscopy. We found that the two processes (i.e., the interdigitation and dehydration of the DPPC bilayers) occur nonsynchronously at two different CH3CN concentrations (4 and 12 wt %). A detailed submolecular picture for the formation mechanism of the L beta I phase was provided during the L-beta to L beta I phase transition at c(CH3CN) = 4 wt To: the conformation state and the hexagonal packing mode of the lipid acyl chains and the hydration properties of the lipid polar groups do not change, and the only difference is that the formed L beta I phase has a tighter lipid acyl chain packing than that of the Lir phase. When c(CH3CN) > 12 wt %, the added CH3CN molecules selectively dehydrate the interfacial carbonyl groups. Thus, two different kinds of L beta I phases differing only in the hydration states of the interfacial carbonyl groups of phospholipids exist in the c(CH3CN) regions of 5-12 and 13-40 wt To, respectively. The strong ability of acetonitrile to induce interdigitation in the lipid bilayers has been discussed in the viewpoint of its toxicity.