It was previously shown that in pea (Pisum sativum), rDNA repeats contain a polar replication fork barrier that blocks progression of the replication machinery moving in the direction opposite to transcription. This barrier maps in the untranscribed spacer close to the 3' end of the 25S gene. Very similar barriers are also found in the rDNA of yeast, Xenopus and mammalian cultured cells. This high conservation indicates that the rDNA barrier plays a relevant biological role. Progression of replication forks through the DNA sequence where the barrier maps in pea was investigated in plasmids replicating in Escherichia coli and Saccharomyces cerevisiae. No barrier was detected in these heterologous systems, indicating that the DNA sequence by itself was insufficient to block the replication machinery. Therefore, trans-acting factors were likely to be required. Taking advantage of the natural sequence heterogeneity in pea rDNA, we obtained evidence that a 27 bp imperfect tandem repeat is involved in the arrest of replication. Moreover, nuclear protein(s) specifically bound to this repeat suggesting that this DNA/protein complex is responsible for the polar arrest of replication forks.