The origin of membraneous structures in the cytoplasm of human erythrocytes infected with the malaria parasite, Plasmodium falciparum, was determined by confocal fluorescence imaging microscopy. When infectious merozoites invaded erythrocytes labeled with the fluorescent, lipophilic, non-exchangeable molecules DiIC16 or DiOC16, a ring of fluorescence was observed surrounding the internal parasite, indicating that the parasitophorous vacuolar membrane (PVM) is formed in part from the erythrocyte membrane. As the parasites matured, fluorescent vesicles were seen to be exported into the erythrocyte cytoplasm, beginning at 6 h post-invasion. During intraerythrocytic development, these dyes were transferred from the PVM to the parasite. When fluorescently labeled merozoites were released from these cells and invaded unlabeled erythrocytes, fluorescence was confined to the parasite throughout the entire erythrocytic cycle. Taken together, these results demonstrate that all vesicles/membranous compartments in the erythrocyte cytoplasm of parasitized erythrocytes (IRBC) contain membrane derived from the PVM. Based on this information, we define pathways that the parasite utilizes to export proteins and lipids to the host cell cytoplasm and surface membrane. When IRBC were labeled post-invasion with DiIC16 or DiOC16 and the parasites allowed to mature for one life cycle, the dyes were confined to the erythrocyte membrane, demonstrating that the host cell membrane of IRBC does not endocytose and there is no membrane exchange from the erythrocyte to the parasite. This investigation helps to resolve two long-standing controversies and provides new insights into the transport pathways that malaria parasites utilize during their development within host erythrocytes.