SUMMARYMost animal transcription factors are categorized as activators or repressors without specifying their mechanisms of action. Defining their specific roles is critical for deciphering the logic of transcriptional regulation and predicting the function of regulatory sequences. Here, we define the kinetic roles of three activating transcription factors in the Drosophila embryo—Zelda, Bicoid and Stat92E—by introducing their binding sites into theeven skippedstripe 2 enhancer and measuring transcriptional output with live imaging. We find that these transcription factors act on different subsets of kinetic parameters, and these subsets can change over the course of nuclear cycle (NC) 14. These transcription factors all increase the fraction of active nuclei. Zelda dramatically shortens the time interval between the start of NC 14 and initial activation, and Stat92E increases the duration of active transcription intervals throughout NC 14. Zelda also decreases the time intervals between instances of active transcription early in NC 14, while Stat92E does so later. Different transcription factors therefore play distinct kinetic roles in activating transcription; this has consequences for understanding both regulatory DNA sequences as well as the biochemical function of transcription factors.