Identification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication.