Malaria infection in humans, caused by single-celled parasites from the genus Plasmodium, is a major global health challenge. Despite marked progress in the last 15 years, more than 400 million deaths occur worldwide annually, the majority being children under age 5. Recent licensure of the first ever malaria vaccine heralds a new era in efforts to control malaria, but the relatively modest efficacy of the RTS,S vaccine means that complementary approaches will be essential if the WHO's goal of a 90% reduction in rates by 2030 is to be realized. Malaria parasites are motile throughout their complex human and mosquito lifecycle. They move by a process called gliding motility, which underpins their ability to reach, cross, and enter host tissues and cells. Gliding is powered by a parasite actomyosin motor the disruption of which kills the infectious parasite. Towards development of the parasite actomyosin motor as a druggable target, our collaborative team has worked to characterize the essential class XIV single-headed myosin motor PfMyoA, the core of gliding motility. We were the first to characterize and crystallize PfMyoA, demonstrating that its function is uniquely tuned by N-terminal heavy chain phosphorylation. We were the first to show the essential role of PfMyoA and its essential light chain in powering red blood cell (RBC) invasion, the stage responsible for all malaria pathogenesis. We have since used PfMyoA mutants to reveal the energetic barriers necessary for RBC invasion using live cell imaging. These foundations expertly position our team to extend investigation of gliding motility across the malaria lifecycle and explore additional Plasmodium myosins and their cellular roles, which are the combined aims of this competitive renewal. We propose (Aim 1) to define the cellular roles of PfMyoB versus PfMyoA by comparing structures, functional properties, and the role of heavy chain phosphorylation in vitro and in vivo. Aim 2 proposes to determine the binding pocket, mechanism of action, and impact on the parasite of two first-in-class small molecule inhibitors of PfMyoA ATPase activity. Aim 3 investigates two other essential Plasmodium myosins (PfMyoF and K), which are virtually unstudied both as motors and potential future druggable targets. PfMyoF likely plays a role as a processive transporter, while PfMyoK likely functions during sexual development, with motor domain inserts typical of reverse-directionality in eukaryotic class VI motors. We will use an integrative approach highlighting in vitro functional assays (motility and ensemble force assays, optical trap assays, steady- state and transient kinetics) and structural studies (X-ray crystallography and cryo-EM) together with live cell approaches (including super resolution imaging) and genetic investigation of motors (conditional knockouts/substitutions) in several stages of the Plasmodium parasite lifecycle. At completion we will have developed a previously unattainable depth o...