The Notch intracellular region (RAMIC) interacts with a DNA binding protein RBP-J to activate transcription of genes that inhibit cell differentiation. The RAM domain and ankyrin (ANK) repeats of mouse Notch1 RAMIC were shown to be responsible for RBP-J binding and necessary for transactivation. The C-terminal portion of Notch1 RAMIC has also been suggested to be important for transactivation. Using GAL4 fusion constructs, we identified a novel transactivation domain (TAD) between the ANK repeats and the PEST sequence of mouse Notch1. The C-terminal half of mouse Notch2 RAMIC also exhibited TAD activity. Unexpectedly, the RBP-J chimeric protein with the Notch1 TAD failed to activate transcription but the activity was recovered by addition of either the RAM domain or ANK repeats. The results suggest that the activity of Notch1 TAD is repressed by fusion with RBP-J because of the presence of a RBP-J-associated co-repressor(s), which could be displaced by either the RAM domain or ANK repeats. Taken together, mouse Notch1 RAMIC can experimentally be separated into three functional domains: the RAM domain and ANK repeats for RBP-J binding and co-repressor displacement and the C-terminal TAD.