DmTTF is a Drosophila melanogaster mitochondrial DNA-binding protein which binds specifically to two homologous non-coding sequences located at the 3' ends of blocks of genes encoded on opposite strands. In order to test whether this protein acts as transcription termination factor, we assayed the capacity of DmTTF to arrest in vitro the transcription catalyzed by mitochondrial and bacteriophage RNA polymerases. Experiments with human S-100 extracts showed that DmTTF is able to arrest the transcription catalyzed by human mitochondrial RNA polymerase bidirectionally, independently of the orientation of the protein-DNA complex. On the contrary when T3 or T7 RNA polymerases were used, we found that DmTTF prevalently arrests transcription when the DNA-binding site was placed in the reverse orientation with respect to the incoming enzymes. These results demonstrate that DmTTF is a transcription termination factor with a biased polarity and suggest that the DNA-bound protein is structurally asymmetrical, exposing two different faces to RNA polymerases travelling on opposite directions. © 2005 Elsevier Inc. All rights reserved.