SummaryIn multicellular animals, the first major event after fertilization is the switch from maternal to zygotic control of development. During this transition, zygotic gene transcription is broadly activated in an otherwise quiescent genome in a process known as zygotic genome activation (ZGA). In fast developing embryos, ZGA often overlaps with the slowing of initially synchronous cell divisions at the mid-blastula transition (MBT). Initial studies of the MBT led to the nuclear-to-cytoplasmic ratio model where MBT timing is regulated by the exponentially increasing amounts of some nuclear component ‘N’ titrated against a fixed cytoplasmic component ‘C’. However, more recent experiments have been interpreted to suggest that ZGA is independent of the N/C ratio. To determine the role of the N/C ratio in ZGA, we generatedXenopusfrog embryos with ∼3-fold differences in genomic DNA (i.e., “N”) by usingX. tropicalissperm to fertilizeX. laeviseggs with or without their maternal genome. Resulting embryos have otherwise identicalX. tropicalisgenome template amounts, embryo sizes, andX. laevismaternal environments. We used theX. tropicalispaternally derived mRNA to identify a high confidence set of exclusively zygotic transcripts. Both ZGA and the increase in cell cycle duration are delayed in embryos with ∼3-fold less DNA per cell. Thus, DNA is an important component of the N/C ratio, which is indeed a critical regulator of zygotic genome activation inXenopusembryos.