Iron is an essential element because it is a key constituent of the metalloproteins involved in cellular respiration and oxygen transport. There is no known regulated excretory mechanism for iron, and homeostasis is tightly controlled via its uptake from the diet. This study assessed in vivo intestinal iron uptake and in vitro iron absorption in a marine teleost, the European flounder Platichthys flesus. Ferric iron, in the form 59FeCl3, was reduced to Fe2+ by ascorbate, and the bioavailability of Fe3+ and Fe2+ were compared. In vivo Fe2+ uptake was significantly greater than Fe3+ uptake and was reduced by the iron chelator desferrioxamine. Fe2+ was also more bioavailable than Fe3+ in in vitro studies that assessed the temporal pattern and concentration-dependency of iron absorption. The posterior region, when compared with the anterior and mid regions of the intestine, was the preferential site for Fe2+ uptake in vivo. In vitro iron absorption was upregulated in the posterior intestine in response to prior haemoglobin depletion of the fish, and the transport showed a Q10 value of 1.94. Iron absorption in the other segments of the intestine did not correlate with haematocrit, and Q10 values were lower. Manipulation of the luminal pH had no effect on in vitro iron absorption. The present study demonstrates that a marine teleost absorbs Fe2+ preferentially in the posterior intestine. This occurs in spite of extremely high luminal bicarbonate concentrations recorded in vivo, which may be expected to reduce the bioavailability of divalent cations as a result of the precipitation as carbonates (e.g. FeCO3).