A direct illumination by focused femtosecond pulses from a near-infrared Ti:sapphire laser in nanojoules peak-energy regime results in a highly localized removal of phospholipids from a fluid, supported lipid bilayer submerged in aqueous media. Lipid-free gaps created in this manner exhibit long-term stability and interrupt membrane fluidity, thereby providing diffusional barriers within the membrane environment. Re-illuminating these lipid-free barriers at comparable peak energies but at higher repetition (similar to 200x) rapidly and selectively erases them. This in situ, reversible, maskless, multiphoton membrane photolithography provides a new means to compartmentalize and regulate membrane fluidity by erecting and erasing diffusional barriers at time scales faster than diffusional time scales. Such an ability to dynamically manipulate membrane diffusional properties should prove useful in designing synthetic models for studies of thermodynamically uphill processes including directed-molecular transport, compositional and free-energy gradients, and nonequilibrium fluctuations in biological membranes.