Based on the hypothesis that Fe+3 must be reduced before Fe absorption, we investigated luminal factors that might participate in the physiological Fe+3 reduction. Rats were fed diets containing 7 mg Fe/kg diet [adequate iron (+Fe]) for 3 wk prior to a 10-min test of 59Fe absorption from an in vivo ligated duodenal segment. During absorption of 59Fe, the oxidation-reduction potential became more reducing and the pH rose in segment contents. There were small but significant differences between the -Fe and +Fe rats. In one experiment, the lumen environment was modified by bile duct ligation and/or intestinal perfusion prior to the absorption test. Ascorbic acid, nonprotein sulfhydryl compounds, Fe+2 and total ionizable Fe were measured in luminal contents. Nonprotein sulfhydryl concentration was positively correlated with, and the best predictor of, Fe absorption in -Fe rats.