Abstract Apicomplexa parasites contribute significantly to human disease burden, including ~1/3 of human populations permanently infected with Toxoplasma gondii. Existing treatments are limited and often toxic to the most affected population of immunocompromised patients. We need a profound knowledge of parasite biology to develop efficient anti-parasitic drugs. Our group focuses on the studies of cell cycle mechanisms that are central to parasite survival and offer a wealth of druggable targets. The cell cycle program orchestrates cell division and ensures the inheritance of the genetic material. Apicomplexan cell cycles are strikingly different from the cell cycles of their hosts. Although T. gondii tachyzoite divides by endodyogeny that resembles a binary division of the conventional eukaryotes, there are substantial differences in cell cycle organization and regulation. It includes the atypical S-phase of Toxoplasma endodyogeny, which is a primary focus of our study. The need for appropriate tools to examine the intricacy of the apicomplexan cell cycle and unconventional regulators significantly impedes the related studies. To fill a major gap in our knowledge of the essential biology of apicomplexan parasites and boost the Toxoplasma cell cycle studies, we engineered a new Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probe. In the Aim 1 experiments, we will test the hypothesis that Toxoplasma endodyogenic cell cycle includes a composite S/M/C phase that runs for nearly half of the division cycle. Using our new ToxoFUCCI probes, we will determine how the intertwined S/M/C phase is organized. In Aim 2, we will determine the mechanism of the S-phase regulation. Designed experiments will test the hypothesis that, contrary to conventional S-phase cyclin dependent kinase (Cdk), controlled release of the sequestrated Cdk-related kinase TgCrk5 regulates DNA replication in the tachyzoites. Using the conditional expression model of TgCrk5, we will determine the functions of the sequestered and the nuclear TgCrk5 and identify the TgCrk5 substrates. Given that T. gondii lacks conventional S-Cdk substrates, we expect to discover a novel TgCrk5 network. The project will advance our knowledge of the fundamental process of parasite survival and have a high potential to discover future efficient drug targets.