Using the atomic force microscope (AFM) in situ, we have demonstrated that acyl chain interdigitation can be induced reversibly by alcohol in supported unilamellar phospholipid bilayers. At alcohol concentrations considerably lower than the critical values determined by other experimental techniques, it was found that interdigitated domains can be induced and these domains were stable over a long period of time. The mechanism of such domain formation remains to be elucidated. This work also serves as an example to illustrate the uniqueness of the AFM as a powerful tool in the study of membrane structure and conformation under physiological conditions at high spatial resolution.