We have employed genetic and two-dimensional (2D) gel electrophoretic methods to identify replication initiation, pausing, and termination sites in the tandem ribosomal DNA (rDNA) repeats of the fission yeast, Schizosaccharomyces pombe. An autonomously replicating sequence (ARS) element, ars3001, maps to a 2.3-kb restriction fragment spanning the junction between the nontranscribed spacer (NTS) and the external transcribed spacer upstream of the ribosomal RNA genes, and 2D gel analysis shows that replication initiates in the NTS portion of the same fragment. A pause region at the 3' end of the rRNA genes inhibits forks from entering these genes counter to the direction of transcription. Thus, most forks move through the genes in the same direction as transcription. In these respects, fission yeast rDNA replication resembles that in the budding yeast, Saccharomyces cerevisiae, and in multicellular eukaryotic organisms. A feature which, so far, has been detected only in fission yeast is the pausing of replication forks in a broad region near the 5.8S rRNA gene.