The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the α-subunit of eukaryotic protein synthesis initiation factor 2 (elF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte elF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, Indicating that protein phosphorylation may play an important role in viral RNA replication. © 1985.