To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucylamido-(4-guanidino)-butane (E-64) sufficient for the needs of the parasite (1). Second, the hemoglobin content of infected erythrocytes decreases 25-75% during the life cycle of erythrocytic parasites (2,3), and the concentration of free amino acids is greater in infected erythrocytes than in uninfected erythrocytes (4). Third, the composition of the amino acid pool of infected erythrocytes is similar to the amino acid composition of hemoglobin (5-7). Fourth, the infection of erythrocytes containing radiolabeled hemoglobin is followed by the appearance of labeled amino acids in parasite proteins (8-10).Hemoglobin degradation occurs predominantly during the trophozoite stage ofthe erythrocytic life cycle ofP. falciparum. Trophozoites ingest erythrocyte cytoplasm and transport it within vesicles to a large central food vacuole (1 1, 12), where the hemoglobin-rich cytoplasm is degraded. In the food vacuole the heme moiety precipitates and is a major component of malarial pigment (13), and globin is hydrolyzed to its constituent free amino acids. The food vacuole of P. falciparum is an acidic (14, 15), membrane-bound (1 1) compartment where proteins are degraded, and therefore it appears to be analogous to the secondary lysosomes of mammalian cells, where multiple proteinases hydrolyze proteins at acid pH (16,17).The enzymatic mechanism of globin degradation within the malarial food vacuole is unknown. In previous studies, aspartic proteinases that degraded denatured hemoglobin were isolated from malaria parasites (18-20). However, denatured hemoglobin is a nonspecific substrate that can be hydrolyzed by many proteinases, and the biological role of the aspartic proteinases cannot be determined from these studies. To determine the enzymatic mechanism of globin degradation in the trophozoite food vacuole we first studied the effects of class-specific proteinase inhibitors on intact parasites. We found that two peptide inhibitors of cysteine proteinases blocked globin degradation in the food vacuole of P. falciparum trophozoites. We also identified a cysteine proteinase of trophozoites that was inhibited by the same two proteinase inhibitors and had biochemical properties that were similar to those of the lysosomal cysteine proteinase cathepsin L. Our results suggest that the cysteine proteinase we identified has a critical role in hemoglobin degradation in the food vacuole of P. falciparum trophozoites.