Atomic Force Microscopy (AFM) has been used to investigate the in situ phospholipase A₂ (PLA₂) degradation of one-component and two-component phospholipid mica supported bilayers composed of dipalmitoylphosphocholine (DPPC) and dimyristoylphosphocholine-distearoylphosphocholine (DMPC-DSPC). The solid supported phospholipid bilayers were imaged in contact mode AFM. Hydrolysis of the lipid bilayers was initiated by injection of PLA₂ into the fluid cell of the atomic force microscope and the enzymatic degradation was monitored in situ at room temperature. The AFM pictures show that the PLA₂ hydrolysis displays an increased activity towards certain preexisting interfacial regions in the one-component DPPC bilayer due to preformed holes appearing in the solid supported lipid film. It is found that the PLA₂ hydrolysis activity, determined by the amount of lipid bilayer disappearing from the mica substrate, varies almost linearly as a function of time elapsed after addition of PLA₂. For the equimolar DMPC-DSPC lipid bilayer mixture, an increased PLA₂ activity is observed towards DMPC enriched small-scale lipid structures.