Replication of the approximately 200 tandem copies of yeast ribosomal RNA genes (rDNA) is known to be initiated within a subset of the repeats, with transcription continuing during the replication process. To examine replication fork movement in this gene cluster, we used a two-dimensional (2D) agarose gel electrophoresis procedure that distinguishes molecules with different branched structures. Replication forks move through most of the rDNA in the same direction in which RNA polymerase I transcribes the 35S rRNA precursor: the 3' end of this transcription unit acts as a barrier to replication forks moving in the direction opposite to RNA polymerase I. The replication fork barrier (RFB) is observed as the accumulation of branched intermediates of specific size. We propose that the act of transcription may influence the movement of replication forks, creating barriers at the 3' ends of actively transcribed genes.