Fluorescence recovery after photobleaching (FRAP) is a classic technique for measurement of the translational diffusion of fluorophores and fluorescently labeled macromolecules. In spot photobleaching, a brief intense light pulse irreversibly bleaches fluorophores in a defined volume of a fluorescent sample. With an attenuated probe beam, the diffusion of unbleached fluorophores into the bleached volume is measured as a quantitative index of fluorophore translational diffusion. A variety of optical configurations, detection strategies, and analysis methods have been used to quantify diffusive phenomena in FRAP measurements. In living cells, FRAP has been used widely for determination of solute/macromolecule diffusion in membranes and aqueous compartments, as well as for semiquantitative analysis of protein transport mechanisms. The introduction of green fluorescent protein (GFP) and other novel cellular labels has motivated a renewed interest in developing quantitative measurement and analysis methods to follow the diffusive and directed transport of defined targets in cells.