Iron chelators are important tools in biochemical studies of iron metabolism and in the therapy of iron overload diseases. Their mode of action is comprised of entry into cells and scavenging intracellular metal, which includes complexation and egress of the complex. Iron is a metal which appears in the cells in various chemical forms and in different compartments. The form of the metal directly affected by the chelators is the most labile, low-molecular-weight type, which is present in the cytosol and is known as the "chelatable iron." This form is thought to be in dynamic equilibrium with several sequestered forms present in the cell, including the iron-responsive proteins. We recently introduced a fluorescent method for assessing the chelatable iron pool of cells, based on the quenching of the fluorescent calcein by metal ions (Breuer et al., J. Biol. Chem., 1995, 270, 24209-24215). In this work we adapted the method for dynamic assessment of chelator efficacy in scavenging iron from cells. In assay 1, red blood cells ghosts are used as a cell membrane model. The free-acid (impermeant) form of calcein is loaded into ghosts by encapsulation (lysis and resealing) and its fluorescence is quenched by addition of permeant iron(II). Chelators added to ghosts lead to iron removal from calcein and hence to recovery of fluorescence, commensurate with their permeation into ghosts and iron binding affinity. In assay 2, human K562 erythroleukemia cells are loaded with calcein via its permeating and cleavable acetoxymethyl form. A fraction of the intracellular calcein fluorescence is quenched in situ by endogenous cellular iron. The rate of dequenching which is obtained after addition of a chelator provides a measure for the scavenging of the intracellular metal, a process which, as in assay 1, depends on chelator permeation and binding affinity for iron. The two methods provide convenient means for assessing the efficacy of candidate chelator structures in depleting cell iron pools. They are also potentially applicable to chelation of other metals such as Co(II), Ni(II), and Cu(II) and to any cellular system or membrane vesicles.