VPg is a virus-encoded protein covalently attached to the 5' end of poliovirus virion RNA. We have used antibody prepared against chemically synthesized VPg to detect two forms of VPg in infected cells. Both forms were specifically immunoprecipitated from lysates of infected cells labeled with [3H]-leucine. One appears to be unmodified VPg because it had the same electrophoretic mobility as synthetic VPg. The other had a larger apparent molecular weight than VPg and could be labeled in vivo with 32Pi. Its structure is VPg-pUpU, the UMP dinucleotide being attached to VPg via a phosphodiester bond to tyrosine, the third amino acid from the NH2 terminus of VPg. This structure is identical to that found at the 5' end of virion and minus-strand RNA.