AbstractMotility of the apicomplexan parasitePlasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone fromPlasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexanToxoplasma gondiidoes not functionally substitute. We expressed a functional full-length PfMyoA with boundmyosintailinteractingprotein (MTIP), the only known light chain of PfMyoA. We then identified an additional “essential” light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 μm/sec, more than twice that of PfMyoA-MTIP (~1.7 μm/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressedP. falciparumactin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.SignificanceMotility of the apicomplexan parasitePlasmodium falciparum, the causative agent of malaria, relies on a divergent actomyosin system powered by the class XIV myosin, PfMyoA. We show that functional PfMyoA can be expressed inSf9cells if aPlasmodium spp. myosin chaperone is co-expressed. We identified an “essential” light chain (PfELC) that binds to PfMyoA in parasites.In vitroexpression of PfMyoA heavy chain with PfELC and the known light chain MTIP produced the fastest speeds of actin movement (~3.8 μm/sec). Duty ratio estimates suggest that ~6 PfMyoA motors can move actin at maximal speed, a feature that may facilitate interaction with short, dynamicPlasmodiumactin filaments. Our findings enable drug screening for myosin-based inhibitors ofPlasmodiumcellular invasion.