We have previously shown that the spatial dynamics of GFP-tagged proteins can be investigated by locally enhancing the fluorescence of GFP using a short UV-laser pulse. 1 We have since developed photobleaching and photoenhancement methods to study binding parameters of GFP-tagged protein domains in intact cells. Here we present an analysis procedure which uses either photobleaching or photoenhancement measurements to determine simultaneously (1) the lateral diffusion coefficient and (2) the dissociation time constant defined for a plasma membrane associated, GFP-tagged protein. The dissociation time constant is defined as the time required for the dissociation of 63% of bound protein and is a functionally relevant parameter related to the binding strength. High affinity interactions are associated with a slow dissociation time constant and low affinity interactions with a fast dissociation time constant. The dissociation time constant of a signaling protein also imposes a critical limit on the cellular response time for signal transduction.