Abstract: Malaria parasites utilize a N-terminal motif termed the Plasmodium export element (PEXEL) to export proteins to the host erythrocyte during blood stage infection. Insight into this signal-mediated trafficking was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two exported proteins that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this N-terminal processing may occur in all exported proteins. PEXEL processing was established to occur upstream of the brefeldin A-sensitive step in the P. falciparum secretory pathway suggesting this N-terminal processing occurs in the parasite endoplasmic reticulum. Furthermore, it was shown that recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein export. Our understanding of PEXEL processing has allowed us to propose that the PEXEL may be defined as a novel signal peptidase cleavage site and a classical N-acetyltransferase substrate sequence. Thus, the putative signal peptidase complex (SPC) subunits of Plasmodium falciparum were expressed in Escherichia coli as N-terminal maltose-binding protein fusions (MBP-P fSPC21 and MBP-PfSPC22/23 1-138) and purified. A fluorogenic substrate based on the PEXEL cleavage site of PfHRPII was synthesized to assess the proteolytic activity of these P. falciparum SPC subunits in vitro. It was revealed that both constructs displayed separate proteolytic activities against the fluorogenic substrate with optimal activity in the physiological pH range. Mass spectrometry analysis of the peptide fragments generated by MBP-PfSPC21, MBP-PfSPC22/23 1-138, and a Triton X-100-soluble parasite extract showed all three proteolytic activities had similar substrate cleavage profiles suggesting that P. falciparum SPC activity is present in the Triton X-100-soluble parasite fraction. The SPC subunits show overlapping substrate specificities that may indicate functional redundancy. Determination of the kinetic parameters for MBP-PfSPC21 and MBP-P fSPC22/23 1-138 reveal an order of magnitude difference between their k_cat/K_m values. Inhibitor profiles demonstrate that PfSPC21 may be responsible for the proteolytic activity detected in the Triton X-100-soluble parasite extract and, therefore, this subunit may be an integral membrane protein. The inhibition profiles for MBP-P fSPC21 and MBP-PfSPC22/23 1-138 suggest they are both serine proteases that utilize different catalytic residues.