Three questions central to understanding the initiation of DNA replication in eukaryotes are: (1) Does DNA synthesis begin at a defined place? (2) What determines replication initiation sites? (3) What regulates an origin to fire only once per cell cycle? A key player in this is the origin recognition complex (ORC), required for assembly of the pre-replication complex (pre-RC), that is converted later to the initiation complex (IC). In both yeast ARS1 and DNA puff II/9A of the metazoan fly Sciara, there is a defined start site of replication adjacent to an ORC-binding site. Although ORC has some inherent preference for certain DNA sequences, other factors may also modulate its binding to DNA. The preferred site where DNA synthesis starts at Sciara II/9A and the boundaries of the initiation zone change during development, when DNA puff amplification occurs. The position of the initiation zone may be influenced by the transcriptional machinery and/or chromatin structure. With regard to the third question, rereplication of the whole genome in yeast occurs when components of the pre-RC are stabilized by mutation. In contrast, a locus-specific amplification factor probably exists to account for site-specific DNA amplification in flies. (C) 2002 Elsevier Science (USA). All rights reserved.