Phosphate compounds are among the factors considered to be most inhibitory to gastrointestinal iron absorption. In this paper the depressant action of phosphate salts and phosphate-containing foods on iron absorption was measured in vivo and compared to the interactions observed between iron and phosphate compounds under conditions similar to those in the gastrointestinal tract. Phosphoproteins or insoluble calcium phosphate bound iron more effectively than did soluble phosphate salts; in the presence of phosphoprotein, iron was not dissolved even at pH 1.3, approximately the acidity of fasting gastric juice, unless ascorbic acid was present. Evidence is present that phosphates act by accelerating the oxidation of ferrous iron to the ferric state and sequestering it as ferric phosphate, a form in which iron is not dialyzable at pH 6 to 7 even in the presence of ascorbic acid. The studies suggest that iron is absorbed only as the free ferrous ion or as low molecular weight complexes capable of passing through a membrane by simple diffusion. The pH of the lumen at the site of absorption is probably between 6 and 7.4. The mixture of foodstuffs encountered by iron in the lumen of both the stomach and upper small intestine is apparently of greater importance than the form in which iron is consumed. Some implications for oral iron therapy are presented.