In mammals, sequence-specific termination of DNA replication within the ribosomal RNA genes is catalyzed by a defined DNA-protein complex that includes transcription termination factor I (TTF-I). Here we show that TTF-I acts as a polar contrahelicase contrary to the intrinsic 3' -->5' helicase activity of SV40 large T antigen. The contrahelicase activity requires binding of TTF-I to its cognate recognition site and the presence of an auxiliary GC-rich sequence, which is able to form a specific secondary structure. Mutations in the GC-rich sequence lead to a loss of folding into correct secondary structure and abrogate contrahelicase activity. The finding suggests that a specific interaction between the Sal box-bound TTF-I and the GC-rich sequence is essential for the inhibition of T antigen helicase. Analyses of N-terminally truncated mutants of TTF-I showed inhibition of helicase by the same domain of TTF-I, which is also responsible for replication fork arrest.