ABSTRACT The process of polytenization plays a crucial role in Drosophila development, and most of the larval tissues are polytene. By analyzing the pattern of DNA replication in embryos pulse-labeled with BrdU, we show that many larval tissues undergo a transition to begin becoming polytene late in embryogenesis. Our results demonstrate that in these larval tissues polyteny results from a modified cell cycle, the endo cell cycle, in which there is only an S (synthesis) phase and a G (gap) phase. A key regulator of the mitotic cell cycle, the product of the string gene (the Drosophila homologue of cdc25), is not required for the endo cell cycle. The developmental regulation of the endo cell cycle is striking in that tissue-specific domains undergo polytene DNA replication in a dynamic pattern at defined times in embryogenesis. During subsequent rounds of the endo cell cycle in late embryogenesis and first instar larval development, the domains are subdivided and the temporal control is not as rigid. The length of the G phase varies among different tissues. By quantifying DNA content, we show that during the early polytene S phases the genome is not fully duplicated.