Merozoites of the malaria parasite Plasmodium falciparum possess on their surface proteolytically processed fragments of the merozoite surface protein-1 (MSP1). Secondary processing of one of these fragments, MSP1(42), always occurs prior to, or at the point of successful erythrocyte reinvasion. It is shown that a product of this secondary processing, MSP1(33), is shed in the form of a noncovalently-associated complex with a number of other proteins, including the MSP1-derived species MSP1(38) and MSP1(83). Secondary processing of MSP1(42) is inhibited by the chelating agents ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethyl ether)-tetraacetic acid (EGTA), and this inhibition is reversible by addition of excess calcium. Secondary processing occurs in preparations of washed, disrupted merozoites, and is inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), indicating that the protease responsible is a membrane-associated serine protease.