Termination of RNA polymerase I (Pol I) transcription requires the interaction of a specific DNA binding factor with terminator elements downstream of the pre-rRNA coding region. Both the terminator elements and the respective termination factors are distinct in yeast and mammals, and differences in the mechanism of transcription termination have been postulated. We have compared in vitro transcription termination of yeast and mouse Pol I using both the murine factor TTF-I, and the yeast homolog Reb1p. We show that, similar to TTF-I, Reb1p was sufficient for pausing of Pol I from either species, but was unable to cause release of the nascent transcripts from the paused ternary complex. The deficiency of Reb1p to mediate transcript release from Pol I of either species was complemented by the recently characterized murine release factor. Thus, both yeast and mouse Pol I termination requires a trans-acting factor that, in conjunction with the T-rich flanking sequence, releases the transcripts and Pol I from the template. The observation that the murine factor causes dissociation of ternary transcription complexes arrested by Reb1p suggests that the mechanism of Pol I termination is highly conserved from yeast to mammals.