DNA sequences and protein factors directing termination of mouse rDNA transcription in a nuclear extract system were examined. Termination is specific and requires a sequence element AGGTCGACCAGATTANTCCG (the Sall box) that is present eight times in the spacer region downstream of the 3' end of pre-rRNA. Exonuclease III protection experiments reveal the binding of a nuclear protein to the Sall box. Deletions, insertions, and point mutations in the Sall box reduce or abolish the interaction with the nuclear factor and disrupt transcription termination. A synthetic oligonucleotide corresponding to the Sall box consensus sequence governs transcription termination in vitro, although with reduced activity. Therefore, other sequences normally surrounding the Sall box appear to contribute to the accuracy and efficiency of termination.