Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actinindependent process.