The small protein (VPg) covalently linked to the 5' end of poliovirus Type 1 (PV-1) RNA has been labeled in vitro with 125I using the Bolton and Hunter reagent. The RNA is not degraded under the conditions used and nearly all the label enters VPg and not the poly-nucleotide chain. When this 125I-labeled RNA is cleaved with RNase III at low monovalent salt concentrations, one major 125I-labeled fragment, approximately 100 nucleotides long, is produced. The corresponding fragment from similar digests of 32P-labeled RNA has also been identified. The 32P-labeled fragment changes electrophoretic mobility after protease treatment indicating that it contains VPg. Furthermore, the RNase T1 oligonucleotide known to be at the 5' terminus of poliovirus RNA is found in T1 digests of the purified fragment. These results confirm that the fragment is derived from the 5' end of the RNA. This fragment will be useful in studies concerning the initiation of protein synthesis during poliovirus infection.