Two independent two-dimensional (2D) gel methods were used to map an origin of replication that is developmentally regulated by the steroid hormone ecdysone, namely an origin for DNA puff amplification in the fungus fly Sciara coprophila. Initiation of replication was found to occur within a small region of no larger than 6 kb by use of the neutral/neutral 2D gel method. Neutral/alkaline 2D gel analyses support the results of the neutral/neutral 2D gels and further define within the origin region an -l-kb area where the majority of replication initiates. This is the first example of an origin of replication in multicellular eukaryotes that has been mapped by 2D gels to such a small defined region. Moreover, replication can be seen by the neutral/alkaline 2D gel method to proceed bidirectionally outward from this replication origin region. These data are consistent with an onion-skin mechanism whereby multiple rounds of DNA replication initiate at a specific origin of replication for Sciara DNA puff amplification.