We investigated vibrational imaging of phospholipid bilayers with CARS microscopy. Single lipid membranes of supported bilayers, giant unilamellar vesicles and intact erythrocyte membrane are detected with the strong resonant signal of the C–H stretching vibration. In addition, it is shown that the CARS signal field of the lipids near the glass/water interface is amplified through mixing with the back-reflected non-resonant CARS field of the glass coverslip. Furthermore, interference effects between two separate bilayers are observed, allowing intermembrane distances to be determined beyond the diffraction-limited resolution of the microscope. Copyright © 2003 John Wiley & Sons, Ltd.