Erythropoiesis-inhibiting factors (EIF) have been demonstrated in plasma from hypertransfused animals and from polycythemic individuals during periods of hyperoxia, but there is a decided discrepancy in the data published. In the present paper methodologic variations of a bioassay for demonstrating the erythropoiesis-inhibiting factor are discussed. In these studies no inhibitor of erythropoiesis could be demonstrated in plasma from hypoxia-induced polycythemic mice (HPM) on posthypoxic day 5. Injections of RBC or an equal amount of hemolyzed RBC were capable of suppressing the stimulatory effects of ESF, indicating that a red cell constituent may be responsible for the inhibitory effect observed. Transfusion-induced polycythemic mice (TPM) were therefore considered to be less suitable for demonstrating erythropoiesis inhibitors. Our results from testing several doses of a urinary EIF in normal mice, TPM and HPM, indicated that the HPM provided the most sensitive assay system. A similar effect was obtained with hypoxia-induced polycythemic rats. The most marked effect was seen in HPM when the EIF was injected shortly before administering the ESF, while the effect was less pronounced when the EIF was injected 24 hr before or after the ESF.