The developmentally regulated amplification of the Drosophilathird chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-β is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the ∼1.2 kb S18 chorion gene and the 840 bp ori-β. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects,suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-β,thereby confirming the sequence specificity of ori-β. Deletion mapping of ori-β identified two required components: a 140 bp 5′ element and a 226 bp A/T-rich 3′ element called the β-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2)recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satineliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminated foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-β nucleate the formation of a ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon chiffon.