Movement of the malaria parasite into a host erythrocyte during invasion is thought to involve polymerization of parasite actin. We have used F-actin affinity chromatography to isolate actin-binding proteins from Plasmodium knowlesi merozoites, in an attempt to identify proteins responsible for regulating parasite actin polymerization during invasion. Five major proteins, of molecular masses 75, 70, 48, 40 and 34 kDa, were reproducibly eluted from the F-actin columns. The 70 kDa actin-binding protein was identified by tryptic peptide microsequencing as heat shock protein-70 kDa (HSC70); this identification was confirmed by Western blotting with anti-HSC70 antibody, and binding of the protein to ATP-agarose. A doublet of 32/34-kDa proteins coeluted with parasite HSC70 from the F-actin and ATP-agarose columns; a complex of these three proteins was also observed by gel filtration chromatography Highly enriched fractions containing the Plasmodium HSC70/32/34 complex inhibited the polymerization of rabbit skeletal muscle actin, in vitro. This capping activity was calcium-independent, and abrogated by phosphatidylinositol 4,5-bisphosphate. The average length of the actin filaments polymerized in presence of the HSC70/32/34-kDa complex was significantly shorter than in the absence of the complex, consistent with a capping activity. The capping or uncapping of actin filament ends by the HSC70/32/34-kDa complex during invasion could provide a mechanism for localized actin filament growth and movement of the parasite into the host cell.