The malarial parasite Plasmodium falciparum has two nucleosome assembly proteins, PfNapS and PfNapL (Chandra, B. R., Olivieri, A., Silvestrini, F., Alano, P., and Sharma, A. (2005) Mol. Biochem. Parasitol. 142, 237-247). We show that both PfNapS and PfNapL interact with histone oligomers but only PfNapS is able to deposit histones onto DNA. This property of PfNapS is divalent cation-dependent and ATP-independent. Deletion of the terminal subdomains of PfNapS abolishes its nucleosome assembly capabilities, but the truncated protein retains its ability to bind histones. Both PfNapS and PfNapL show binding to the linker histone H1 suggesting their probable role in extraction of H1 from chromatin fibers. Our data suggests distinct sites of interaction for H1 versus H3/H4 on PfNapS. We show that PfNapS and PfNapL are phosphorylated both in vivo and in vitro by casein kinase-II, and this modification is specifically inhibited by heparin. Circular dichroism, fluorescence spectroscopy, and chymotrypsin fingerprinting data together suggest that PfNapL may undergo very small and subtle structural changes upon phosphorylation. Specifically, phosphorylation of PfNapL increases its affinity 3-fold for core histones H3, H4, and for the linker histone H1. Finally, we demonstrate that PfNapS is able to extract histones from both phosphorylated and unphosphorylated PfNapL, potentially for histone deposition onto DNA. Based on these results, we suggest that the P. falciparum NapL is involved in the nucleocytoplasmic relay of histones, whereas PfNapS is likely to be an integral part of the chromatin assembly motors in the parasite nucleus.