Medium conditioned by the monocyte-like cell line GCT contains colony-stimulating activity (CSA), a mediator of in vitro granulopoiesis. Also, the conditioned medium (CM) contains erythroid-enhancing activity (EEA), which can be demonstrated in a system utilizing either nonadherent marrow or blood mononuclear cells, erythropoietin (1-2 units/ml), and 20 ml/dl fetal calf serum. Under these conditions, GCT CM enhances the growth of CFU-E and BFU-E. Attempts were made to characterize the molecular features of EEA. Serum-free GCT cell CM was fractionated on Sephacryl S200 and Ultrogel AcA54. EEA and CSA cochromatographed with apparent molecular weights of approximately 40,000 daltons on Sephacryl and approximately 30,000 daltons on Ultrogel. Fractionation on DEAE Sephacel led to an apparent separation of CSA from EEA; however, when diluted, the fractions containing CSA and EEA. Undiluted fractions containing potent CSA inhibited erythropoiesis; however, dilution of these fractions resulted in marked EEA. Diluted crude GCT CM and DEAE Sephacel fractions enriched in EEA were also capable of sustaining BFU-E in liquid culture and mediating erythropoietin-independent colony growth. CSA could not be unequivocally separated from EEA on concanavalin A-Sepharose, since the diluted void volume containing CSA also had EEA. EEA was present in CM boiled for 60 minutes, whereas CSA was markedly reduced but not abolished. The inverse relationship between CSA concentration and EEA mandates dilution of fractions when bioassayed for these two activities. Although CSA and EEA are similar in molecular weight, they appear to be partially separable by ion-exchange chromatography and heat stability.