Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin Hp and ceruloplasmin (Cp), respectively. In swine copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced as evidenced by the loss of copper-dependent plasma ferroxidase I activity and in enterocytes reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced and liver iron doubled. CD reduced duodenal mucosal iron and ferritin, while increasing iron absorption. Duodenal mucosal DMT1-IRE and ferroportin expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron deficient-anemic animals strong correlations were found between mucosal iron, ferritin, and iron absorption, suggesting the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages because Kupffer cells of the liver and non-heme iron levels of the spleen were normal during copper deficiency suggesting another role for Cp in these cells.