The digestive vacuole of Plasmodium falciparum is the site of hemoglobin degradation, heme polymerization into crystalline hemozoin, and antimalarial drug accumulation. Antibodies identified histidine-rich protein II (HRP II) in purified digestive vacuoles. Recombinant or native HRP II promoted the formation of hemozoin, and chloroquine inhibited the reaction. The related HRP III also polymerized heme, and an additional HRP was identified in vacuoles. It is proposed that after secretion by the parasite into the host erythrocyte cytosol, HRPs are brought into the acidic digestive vacuole along with hemoglobin. After hemoglobin proteolysis, HRPs bind the liberated heme and mediate hemozoin formation.