An in vitro incubation at pH 2 of EDTA, cysteine, lactic or succinic acids with each of five iron sources, [hydrogen (HRI) and electrolytically reduced elemental iron (ERI), ferric chloride (FeCl3), ferrous sulfate (FeSO4) and ferric orthophosphate (FOP)] at a 10:1 molar ratio (ligand:iron) was evaluated for its effect on iron solubilization in a wheat flake cereal subjected to a sequential gastrointestinal pH treatment from endogenous pH (E) to 2 to 6. Incubation significantly enhanced the iron solubilizing potential of EDTA at each pH with HRI and ERI, while lactic and succinic acids were similarly effective with FeSO4 and FeCl3 at pH 2. The reducing potential of cysteine, along with its role as a ligand, generated substantial amounts of Fe+ 2 (pH 2) at the apparent expense of complexed iron. However, with the exception of ERI (pH E), incubation did not increase cysteine's effectiveness in producing more soluble iron (ionic + complexed). This indicates that pH was the major solubilizing factor. Due to FOP's relative insolubility, incubation proved ineffective in all instances. These in vitro results indicate that acid incubation to form a ligand-iron complex has the potential to improve bioavailability of iron.