In its host erythrocyte, the malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole, the membrane of which forms a barrier between the host cell cytosol and the parasite surface. The vacuole is a unique compartment because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. As a prerequisite for the characterization of the vacuolar proteome, we have developed an experimental approach that allows the selective biotinylation of soluble vacuolar proteins. This approach utilizes nonpermeant biotin derivatives that can be introduced into infected erythrocytes after selective permeabilization of the erythrocyte membrane with the pore-forming protein streptolysin O. The derivatives gain access to the vacuolar lumen but not to the parasite cytosol, thus providing supportive evidence for the existence of nonselective pores within the vacuolar membrane that have been postulated based on electrophysiological studies. Soluble vacuolar proteins that are biotin-labeled can be isolated by affinity chromatography using streptavidin-agarose.