The protein (VPg) covalently attached to the 5' terminus of poliovirus RNA has been reported to resolve into two forms, separable by electrofocusing, yet the viral gene sequence predicts only one apparent gene for VPg. The two VPg species were separated and analyzed for structural differences. The protein contains no phosphorylated amino acid residues other than the junction tyrosine linked to the nucleic acid moiety. After isolation, the acidic form of VPg remains stable, but the more basic form again generates a distribution of both species. This suggests some lability or modification of at least one amino acid residue postsynthesis. The position of the alteration in the protein was localized to the amino-terminal tryptic peptide, containing nine amino acids.