Eukaryotic DNA replication forks stall at natural replication fork barriers or Ter sites located within the ribosomal DNA (rDNA) intergenic spacer regions during unperturbed DNA replication. The rDNA intergenic spacer of the fission yeast Schizosaccharomyces pombe contains four polar or orientation-specific fork barriers, Ter1-3 and RFP4. Whereas the transcription terminator Reb1p binds Ter2 and Ter3 to arrest replication, the factor(s) responsible for fork arrest at Ter1 and RFP4 remain unknown. Using linker scanning mutagenesis, we have narrowed down minimal Ter1 to 21 bp. Sequence analysis revealed the presence of a consensus binding motif for the essential switch-activating and genome-stabilizing protein Sap1p within this region. Recombinant Sap1p bound Ter1 with high specificity, and endogenous Ter1 binding activity contained Sap1p and comigrated with the Sap1p-Ter1 complex. Circular permutation analysis suggested that Sap1p bends Ter1 and SAS1 upon binding. Targeted mutational analysis revealed that Ter1 mutations, which prevent Sap1p binding in vitro, are defective for replication fork arrest in vivo, whereas mutations that do not affect Sap1p binding remain competent to arrest replication. The results confirm the hypothesis that the chromatin organizer Sap1p binds site-specifically to genomic regions other than SAS1 and support the notion that Sap1p binds the rDNA fork barrier Ter1 to cause polar replication fork arrest at this site but not at SAS1.