Two-dimensional fluorescence depolarization measurement was achieved by a fluorescence microscope equipped with polarizing devices and an image intensified CCD camera. Anisotropy images were acquired using living cells stained with a membrane specific binding dye. It was found that the bound dye is spatially distinguishable from the free dye which does not bind to the membrane of a cell, due to the difference in rotational Brownian motion. In addition, we succeeded in obtaining fluorescence depolarization images by means of time- resolved measurement and two-photon excitation measurement. Two-photon excitation images showed a superior signal-to-noise ratio compared to one-photon excitation images. Fluorescence depolarization imaging will therefore prove a powerful tool for studying molecular functions in cells.