To meet the demand for the rapid synthesis of chorion (eggshell) proteins, Drosophila ovarian follicle cells amplify the chromosomal loci containing the chorion gene clusters up to 60-fold. Amplification occurs by repeated firing of one or more origins located within each gene cluster. Deletion analyses of transgenic constructs derived from the third chromosome cluster have identified a 320-bp amplification control element (ACE3) required for amplification, as well as several stimulatory amplification enhancing regions (AERs). Two-dimensional (2D) gel analyses have identified multiple DNA replication initiation sites (origins) that partially overlap in location with ACE3 and the AERs. To further study sequence requirements for amplification, a vector was used in which transgenic constructs are protected from chromosomal position effects by flanking insulator elements, the suppressor Hairy-wing protein binding site (SHWBS). Using the buffered vector, the 320-bp ACE3 and an 884-bp element designated ori-β were found to be necessary and sufficient for amplification. Two-dimensional gels revealed that ori-β was acting as the origin. In contrast, origin activity could not be detected for ACE3. An insulator placed between ACE3 and ori-β inhibited amplification, indicating that ACE3 activates ori-β in cis. The results suggest that ACE3 acts as a replicator and support and extend the replicator model for the organization of metazoan chromosomal replicons.