Secondary processing of the Plasmodium falciparum malaria merozoite surface protein-1 (MSP-1) is defined as a single proteolytic cleavage within the carboxy-terminal membrane-bound component of the MSP-1 protein complex on the free merozoite surface. The N-terminal cleavage product (MSP-1(33)) is shed from the parasite surface along with a number of other polypeptides, whereas the C-terminal processing product remains bound to the merozoite surface and is the only part of MSP-1 detectable in the newly invaded host cell. We report that secondary processing of MSP-1 takes place in a similar manner on invasive merozoites of the simian malaria parasite Plasmodium knowlesi. Processing can take place to a limited extent in pure isolated merozoites; however, within 10 min of the addition of purified invasive merozoites to rhesus erythrocytes, processing and shedding of MSP-1 has gone to completion only in those parasites which have undergone invasion; residual free merozoites remain uniformly reactive with antibodies against MSP-1(33). Successful invasion is therefore associated with complete shedding of MSP-1(33) from the merozoite surface. The nucleotide sequence of the 3' domain of the P. knowlesi MSP-1 gene is also presented.