Previous studies have suggested that iron mucosal protein complexes, possibly related to a carrier substance, are involved in intestinal iron transport. Since there is evidence indicating that phenobarbital induces an increase in intestinal microsomal proteins and enzymes, it was postulated that phenobarbital might affect iron absorption by increasing the synthesis of a carrier protein for iron. This investigation describes studies carried out in the rat to determine the effect of phenobarbital on the absorption of inorganic and hemoglobin iron labeled with Fe 59 . In vivo the absorption of Fe 59 SO 4 was significantly increased in phenobarbital-treated rats compared to controls (20.4 ± 5.7 versus 9.8 ± 4.2%; P P P 0.001). In vitro, phenobarbital treatment resulted in a significant increase in mucosal transfer of Fe 59 SO 4 by everted duodenal gut sacs compared to controls (51.3 ± 8.8. versus 28.0 ± 5.9 mμmoles; P 59 SO 4 by jejunal gut sacs. It is postulated that the phenobarbital-mediated increase in absorption of inorganic iron is related to increased synthesis of a carrier substance that influences iron transport. Further, it is suggested that the increased absorption of hemoglobin iron resulting from phenobarbital treatment is due, at least in part, to increased mucosal heme-splitting enzyme acitivity.