The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine- phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.