Iron chelators belonging to three distinct chemical families were assessed in terms of their physicochemical properties and the kinetics of iron chelation in solution and in two biological systems. Several hydroxypyridinones, reversed siderophores, and desferrioxamine derivatives were selected to cover agents with different iron-binding stoichiometry and geometry and a wide range of lipophilicity, as determined by the octanol-water partition coefficients. The selection also included highly lipophilic chelators with potentially cell-cleavable ester groups that can serve as precursors of hydrophilic and membrane-impermeant chelators. Iron binding was determined by the chelator capacity for restoring the fluorescence of iron-quenched calcein (CA), a dynamic fluorescent metallosensor. The iron-scavenging properties of the chelators were assessed under three different conditions: (a) in solution, by mixing iron salts with free CA; (b) in resealed red cell ghosts, by encapsulation of CA followed by loading with iron; and (c) in human erythroleukemia K562 cells, by loading with the permeant CA-acetomethoxy ester, in situ formation of free CA, and binding of cytosolic labile iron. The time-dependent recovery of fluorescence in the presence of a given chelator provided a continuous measure for the capacity of the chelator to access the iron/CA-containing compartment. The resulting rate constants of fluorescence recovery indicated that chelation in solution was comparable for the members of each family of chelators, whereas chelation in either biological system was largely dictated by the lipophilicity of the free chelator. For example, desferrioxamine was among the fastest and most efficient iron scavengers in solution but was essentially ineffective in either biological system when used at ≤200 μm over a 2-hr period at 37°. On the other hand, the highly lipophilic and potentially cell-cleavable hydroxypyridinones and reversed siderophores were highly efficient in all biological systems tested. It is implied that in K562 cells, hydrolysis of these chelators is relatively slower than their ingress and binding of intracellular iron. The chelator-mediated translocation of iron from cells to medium was assessed in ⁵⁵Fe-transferrin-loaded K562 cells. The speed of iron mobilization by members of the three families of chelators correlated with the lipophilicity of the free ligand or the iron-complexed chelator. The acquired information is of relevance for the design of chelators with improved biological performance.