Iron speciation in rodent diet and rat gastrointestinal tract lumen during dietary digestion and absorption was investigated with a novel selective extraction technique. Five Fe fractions were identified, namely exchangeable (soluble in 1 M-magnesium chloride), carbonate-bound (soluble in mild acid), oxide-bound (soluble in hydroxylamine-acetic acid), organic-bound (soluble after treatment with peroxide in nitric acid) and residual. Fe from the pelleted diet was mobilized by rat stomach to the exchangeable fraction, then redistributed to the carbonate- and oxide-bound fractions on passage through the proximal small intestine. In vitro incubation of diet with hydrochloric acid failed to mimic the in vivo effect of the stomach. In vitro neutralization of stomach contents with bicarbonate was found to produce a similar effect on Fe speciation to that seen when diet passed the proximal small intestine in vivo. Comparison of 59Fe speciation in extrinsically labelled diet with endogenous Fe speciation showed that extrinsic labelling does not uniformly label all endogenous species. The experiments suggest that selective extraction may provide a useful approach to the study of Fe species present in diets, in vitro digestions and gastrointestinal contents.