Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. We have shown previously that the human TATA-binding protein (TBP), a general transcription factor required for transcription of all RNA polymerase II genes, is directly cleaved both in vitro and in vivo by the virus-coded protease 3CPro. 3CPro specifically cleaves glutamine-glycine bonds in the viral polyprotein. Cellular transcription factor TBP contains three glutamine-glycine sites, at amino acids 12, 18, and 108. By using site-directed mutagenesis, we determined that the glutamine-glycine bond at amino acid 18, but not that at amino acid 12 or 108, is cleaved by the viral protease. Both the glutamine and the glycine appear to be important for the cleavage. Further mutations around the glutamine-glycine site at position 18 suggest that determinants other than the glutamine-glycine bond in TBP are also required for 3CPro-induced cleavage. An alanine at position P4 and a proline at position P2, proximal to the scissile glutamine-glycine pair, appear to be important for 3CPro-mediated cleavage of TBP. Our results suggest that the cleavage specificity of 3CPro for a cellular transcription factor is very similar to its mode of cleavage of viral polyproteins.