A confocal time-resolved fluorescence anisotropy imaging set-up is presented. It combines a confocal laser scanning microscope equipped with a pulsed laser and two time gated detection systems with 4 gates each (LiMo, originally developed for FLIM). The anisotropy decays obtained with the time gating system yield results that compare well with the high time-resolution (non-imaging) decays recorded using Time Correlated Single Photon Counting. Time resolved anisotropy imaging experiments on cells expressing GPI-GFP were carried out. Clear distinction could be made between the anisotropy in the plasma membrane and in the interior of the cell.© (2007) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.