Features of iron absorption by the upper small intestine and ileum in the adult guinea-pig have been compared. Autoradiographic methods have revealed that whilst only upper villus enterocytes in the duodenum-jejunum are involved in brush-border uptake, the entire villus epithelium in the ileum displays this function. Consistent with these results was the finding that brush-border membrane vesicles prepared from ileal mucosa demonstrated a higher capacity for iron uptake (expressed per mg protein) compared to vesicles of duodenal origin. Iron transport to blood following 30 min exposure of mucosa to 59Fe-ascorbate was some elevenfold higher in upper, compared to lower, small intestine. The relevance of these findings to our knowledge of the cellular processes involved in intestinal iron absorption and its regional localization is discussed.