Although hemochromatosis and pathological situations due to chronic iron overload have been extensively described, there is little information about the influence of iron on other trace elements in the cell. The aim of this study was to investigate changes in the concentration of zinc, manganese, and copper in the liver, spleen, and brain of rats after iron overload. Iron overload in Wistar rats was achieved by iron-supplemented diet or by intraperitoneal or intravenous injection of polymaltose iron. Iron, zinc, manganese, and copper were determined by atomic absorption spectrophotometry. Iron overload in rats, regardless of the route of its application, resulted in an increase not only of iron but also of zinc and manganese in the liver and the spleen, whereas the content of these metals in the brain did not change. The copper content of the liver, spleen, and brain remained the same after iron overload. The increase of zinc and manganese in the liver and spleen following iron overload was probably a result not only of increased intestinal absorption but also of increased uptake from the cell. This is also supported by the fact that no increase in the zinc and manganese concentrations occurred in the brain since, despite iron overload, the iron content remained constant.