Hemoglobin degradation during the asexual cycle of Plasmodium falciparum is an obligate process for parasite development and survival. It is established that hemoglobin is transported from the host erythrocyte to the parasite digestive vacuole (DV), but this biological process is not well characterized. Three-dimensional reconstructions made from serial thin-section electron micrographs of untreated, trophozoite-stage P. falciparum-infected erythrocytes (IRBC) or IRBC treated with different pharmacological agents provide new insight into the organization and regulation of the hemoglobin transport pathway. Hemoglobin internalization commences with the formation of cytostomes from localized, electron-dense collars at the interface of the parasite plasma and parasitophorous vacuolar membranes. The cytostomal collar does not function as a site of vesicle fission but rather serves to stabilize the maturing cytostome. We provide the first evidence that hemoglobin transport to the DV uses an actin-myosin motor system. Short-lived, hemoglobin-filled vesicles form from the distal end of the cytostomes through actin and dynamin-mediated processes. Results obtained with IRBC treated with N-ethylmaleimide (NEM) suggest that fusion of hemoglobin-containing vesicles with the DV may involve a soluble NEM-sensitive factor attachment protein receptor-dependent mechanism. In this report, we identify new key components of the hemoglobin transport pathway and provide a detailed characterization of its morphological organization and regulation. Malaria is a devastating disease that infects Ͼ300 million people each year, resulting in Ͼ1 million deaths, with the protozoan species Plasmodium falciparum being responsible for the majority of these deaths (1). The morbidity and mortality associated with the disease are largely the result of the parasite's asexual intraerythrocytic cycle (2). P. falciparum digests host cell hemoglobin to support parasite growth and asexual replication during the intraerythrocytic stage (3, 4). In order for the parasite to survive within the erythrocyte host, it degrades approximately 80% of the erythrocyte hemoglobin (5), with the majority of this digestion occurring during the trophozoite stage (18 to 32 h postinvasion) (3). The bulk of hemoglobin degradation occurs via a semiordered process by proteases contained within the parasite's digestive vacuole (DV) (6).The transport of hemoglobin from the host erythrocyte cytosol to the parasite DV is a long-known but poorly understood biological process. The internalization of hemoglobin is thought to occur through an unusual structure, the cytostome. A cytostome is defined as a localized invagination of the parasite's outer membranes (the parasitophorous vacuolar membrane [PVM] and the parasite plasma membrane [PPM]), with a submembranous electron-dense collar associated with the neck of the cytostome (7, 8). It has traditionally been assumed that hemoglobin transport commences with the cytostome pinching off at the neck to form a double-membrane, hemoglobin-f...