S-phase and DNA damage checkpoint controls block the onset of mitosis when DNA is damaged or DNA replication is incomplete. It has been proposed that damaged or incompletely replicated DNA generates structures that are sensed by the checkpoint control pathway, although little is known about the structures and mechanisms involved. Here, we show that the DNA replication initiation proteins Orp1p and Cdc18p are required to induce and maintain the S-phase checkpoint in Schizosaccharomyces pombe. The presence of DNA replication structures correlates with activation of the Cds1p checkpoint protein kinase and the S-phase checkpoint pathway. By contrast, induction of the DNA damage pathway is not dependent on Orp1p or Cdc18p. We propose that the presence of unresolved replication forks, together with Orp1p and Cdc18p, are necessary to activate the Cds1p-dependent S-phase checkpoint.