Male Wistar rats (188 g) were fed on a semi-synthetic (SS) diet (38 mg iron/kg) ad lib. for 7 d and then meal-fed for 1 d. After a 21 h fast each rat was given one meal (10 g) of high-Fe SS (500 mg Fe/kg; high-Fe group) or control (38 mg Fe/kg; control group) diet. After 16 h 2 ml of an 59Fe-labelled ferrous sulphate solution (18 kBq 59Fe; 120 μg Fe) was administrated by gavage and equal numbers of rats from each group were killed 6 or 24 h after dosing. Mucosal uptake of 59Fe from the gut lumen and transfer of 59Fe from mucosa into the carcass were measured. Total Fe content of the duodenum was also determined. Mucosal 59Fe uptake and transfer were markedly lower in the high-Fe group compared with the control group. The Fe content of the duodenum, the major region of Fe absorption, was significantly greater in the high-Fe group than in the controls. A larger amount of Fe may thus have been released into the lumen of the high-Fe rats, via mucosal cell turnover, resulting in a greater lumen dilution of the 59Fe dose in this group compared with the controls. Calculations are presented which demonstrate that such an effect could not possibly account for the observed difference in mucosal 59Fe uptake between groups. Differences in rates of ‘cold’ Fe and 59Fe loss from the duodenal mucosa during the 6–24 h interval suggested that, at the time of dosing, Fe retained in the mucosa from the previous meal had been incorporated into a non-exchangeable pool and as such would not dilute radioactive Fe entering the mucosa. It was concluded that whole-body 59Fe retention from a labelled source, given orally after an overnight fast, provided an accurate estimate of Fe absorption.