Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative 1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.The protozoan, Toxoplasma gondii, is an obligate intracellular parasite that can invade and replicate within any nucleated cell of vertebrate hosts, including humans (1-3). Invasion by T. gondii tachyzoites is mediated by the sequential regulated release of specialized secretory organelles of the parasite including the micronemes, rhoptries, and dense granules (4). In early observations, the penetration of host cells by tachyzoites was enhanced by the addition of partially purified lysosomal enzymes (5). In addition, proteinases have been implicated in host cell invasion in other members of the Apicomplexa such as Plasmodium (6) and Eimeria tenella (7). We now show that a cathepsin B, toxopain-1, is strongly implicated in T. gondii invasion and that infection can be interrupted with specific cathepsin B inhibitors.Another unusual feature of Toxoplasma is the absence of a morphologically identifiable lysosomal system. In higher eukaryotic cells, acidic cathepsins in lysosomes are important in protein processing and breakdown. The mammalian precursor of cathepsin B is targeted to the lysosomal compartment by mannose 6-P for proteolytic activation (8). Thus, the apparent lack of a lysosomal system in Toxoplasma raises a number of questions regarding cellular proteinase functions within the parasite. The rhoptries are club-shaped organelles located at the apical end of the parasites with no known counterpart outside of the phylum Apicomplexa (9). Although nine rhoptry proteins (ROP1-9) 1 have been identified to date (10), a definitive function has only been determined for ROP2, which mediates binding of host mitochondria to the parasitophorous membrane (11). How rho...