To investigate replication fork progression along the tandemly repeated rRNA genes of Xenopus laevis and Xenopus borealis, rDNA replication intermediates from dividing tissue culture cells were analyzed by two-dimensional gel electrophoresis. Analysis of the direction of replication in the rRNA coding regions revealed replication forks moving in both directions. However, in both frog species, polar replication fork barriers (RFB) arresting forks approaching the rRNA transcription units from downstream were identified. Whereas in X. borealis the RFB maps to a defined site close to the transcription terminator, in X. laevis the arrest of fork movement can occur at multiple positions throughout a 3' flanking repetitive spacer region. A short DNA element located near the respective RFB sites is shared between these two related frog species, suggesting its possible involvement in the arrest of replication fork movement. In a subset of rDNA repeats, these barriers cause an absolute block to replication fork progression, defining the sites where replicon fusion occurs, whereas in the remainder repeats, most probably in the non-transcribed gene copies, the replication machinery can pass the RFB sequences and replicate the rRNA transcription unit in a 3'-to-5' direction.