Developmentally regulated initiation of DNA synthesis was studied in the fly Sciara at locus II/9A. PCR analysis of nascent strands revealed an initiation zone that spans approximately 8 kb in mitotic embryonic cells and endoreplicating salivary glands but contracts to 1.2 to 2.0 kb during DNA amplification of DNA puff II/9A. Thus, the amplification origin occurs within the initiation zone used for normal replication. The initiation zone left-hand border is constant, but the right-hand border changes during development. Also, there is a shift in the preferred site for initiation of DNA synthesis during DNA amplification compared to that in preamplification stages. This is the first demonstration that once an initiation zone is defined in embryos, its borders and preferred replication start sites can change during development. Chromatin immunoprecipitation showed that the RNA polymerase II 140-kDa subunit occupies the promoter of gene II/9-1 during DNA amplification, even though intense transcription will not start until the next developmental stage. RNA polymerase II is adjacent to the right-hand border of the initiation zone at DNA amplification but not at preamplification, suggesting that it may influence the position of this border. These findings support a relationship between the transcriptional machinery and establishment of the replication initiation zone.