Recent studies have demonstrated that cholestyramine, an anionic exchange resin, binds inorganic and hemoglobin ironin vitro and inhibits the absorption of these compounds from the rat small intestine. However, these studies did not answer the question: Will prolonged cholestyramine administration cause depletion of iron stores and development of iron deficiency anemia? Accordingly, rats were fed diets of standard rat chow or standard rat chow containing either 2 or 5% cholestyramine for 6 months. In the 5% cholestyramine group the hematocrit and hemoglobin values were significantly lower as compared to controls (37.5 vs 43.6%;P<0.001) and (11.4 vs 14.2 g%;P<0.001). Serum iron values in the 5% cholestyramine groups were significantly less than the control group (158±23 vs 205±15 µg%;P<0.001). In the control group nonheme iron in the liver, spleen and duodenum was 206±31, 1482±212 and 12.5±1.1 µg/g tissue, respectively. In the 2% cholestyramine-treated group, liver nonheme iron was reduced to 172±33 µg/g, spleen nonheme iron to 1266±447 µg/g and duodenal iron to 11.7±1.0 µg/g. In the 5% cholestyramine-treated group liver nonheme iron was significantly reduced to 143±35 µg/g, spleen nonheme iron to 1030±3.4 µg/g and duodenal nonheme iron to 10.2±1.4 µg/g respectively. In addition, a significant decrease in serum vitamin B12 levels was noted in cholestyramine-fed rats compared to controls (256±83 vs 404±118 µµg/ml). These data indicate that prolonged administration of cholestyramine to rats results in significantly decreased serum iron, tissue nonheme iron stores and serum vitamin B12 levels associated with decreased hematocrit and hemoglobin levels.