During erythroid development erythropoietin (EPO) binds specifically to a receptor primarily present on committed erythroid progenitors, stimulating mitogenic, survival, and differentiative growth response pathways. Other modes of erythropoietin receptor (EPO-R) activation, such as interaction with the env gene Friend virus envelope glycoprotein (F-gp55) of spleen focus- forming virus or specific mutations in the extracellular domain of the EPO- R, give rise to pathological consequences, in vivo and EPO-independent proliferation and differentiation of cultured cells. Activating extracellular receptor mutations result in covalently linked receptor homodimers. These observations and others have led to the proposal that EPO activates the EPO- R by inducing dimer formation on the cell surface. It has been assumed that F-gp55 also induces dimer formation of the EPO-R; however, clear evidence of this is lacking. In addition, EPO and F-gp55 stimulation of the EPO-R elicit different biological responses. To probe whether the cell surface EPO-R is structurally different with these activators, we contrasted the cell surface EPO-R complex formed following receptor activation by EPO, F-gp55, and mutations in the extracellular domain of the receptor. Our results indicate that cell surface forms of activated EPO-R differ, as judged by their differential association with F-gp55 and pattern of associated cell surface proteins. Interestingly, we find that the env gene of an anemic strain of Friend virus, Rauscher virus envelope glycoprotein, does not interact with the EPO-R at the cell surface. Thus, the mode of Rauscher virus envelope glycoprotein-induced erythroblastosis may be distinct from F-gp55-induced erythroblastosis and possibly not involve the EPO-R.