In iron overload, non-transferrin-bound iron (NTBI) is found in plasma and is rapidly removed by hepatocytes. Some of this NTBI is excreted into bile. Biliary excretion of NTBI, in the form of an iron-deferiprone chelate, is greatly increased by deferiprone, an iron chelator. The aim of this study was to test whether biliary iron as such or as an iron-deferiprone chelate (both originating from plasma NTBI) is absorbed from the intestine and re-secreted into bile. In healthy biliary fistula (donor) rats, biliary 55Fe originating from plasma NTBI was obtained by injecting Fe citrate (to saturate transferrin) followed by 55Fe. This biliary 55Fe was infused into the duodenum of (recipient) rats whose transferrin was saturated or unsaturated. Similar experiments were performed using iron-overloaded (donor) rats given deferiprone, followed by infusion of the biliary 55Fe-deferiprone chelate into iron-overloaded (recipient) rats. The results show that in healthy (recipient) rats, duodenal infusion of 55Fe from NTBI was followed by increased plasma 55Fe when transferrin was unsaturated, or by biliary excretion of 55Fe when transferrin was saturated, indicating intestinal absorption of 55Fe. In iron-overloaded rats, neither plasma nor bile became radioactive, indicating no intestinal absorption of iron from the deferiprone chelate. We conclude that biliary iron, originating from NTBI, is absorbed from the intestine, and undergoes enterohepatic circulation if transferrin is saturated. In iron-overloaded rats, biliary iron originating from plasma NTBI and present as an iron-deferiprone chelate in bile is not absorbed.