In a rat model of chronic mountain sickness, the excessive polycythemic response to hypoxic exposure is associated with profound splenic erythropoiesis. We studied the uptake and distribution of radioactive iron and red blood cell (RBC) morphology in intact and splenectomized rats over a 30-day hypoxic exposure. Retention of (59)Fe in the plasma was correlated with (59)Fe uptake by both spleen and marrow and the appearance of (59)Fe-labeled RBCs in the blood. (59)Fe uptake in both the spleen and the marrow paralleled the production of nucleated RBCs. Splenic (59)Fe uptake was approximately 10% of the total marrow uptake under normoxic conditions but increased to 60% of the total marrow uptake during hypoxic exposure. Peak splenic (59)Fe uptake and splenomegaly occurred at the most intense phase of erythropoiesis and coincided with the rapid appearance of (59)Fe-labeled RBCs in the blood. The bone marrow remains the most important erythropoietic organ under both resting and stimulated states, but inordinate splenic erythropoiesis in this rat strain accounts in large measure for the excessive polycythemia during the development of chronic mountain sickness in chronic hypoxia.