Termination of transcription by RNA polymerase I (Pol I) is a two-step process which involves pausing of elongating transcription complexes and release of both pre-rRNA and Pol I from the template. In mouse, pausing of elongation complexes is mediated by the transcription termination factor TTF-I bound to the 'Sal box' terminator downstream of the rDNA transcription unit. Dissociation of paused ternary complexes requires a cellular factor, termed PTRF for Pol I and transcript release factor. Here we describe the molecular cloning of a cDNA corresponding to murine PTRF. Recombinant PTRF is capable of dissociating ternary Pol I transcription complexes in vitro as revealed by release of both Pol I and nascent transcripts from the template. Consistent with its function in transcription termination, PTRF interacts with both TTF-I and Pol I. Moreover, we demonstrate specific binding of PTRF to transcripts containing the 3' end of pre-rRNA. Substitution of 3'-terminal uridylates by guanine residues abolishes PTRF binding and impairs release activity. The results reveal a network of protein-protein and protein-nucleic acid interactions that governs termination of Pol I transcription.