The rhoptries of Plasmodium falciparum are formed during a restricted period in the asexual erythrocytic cycle. The steps required for rhoptry biogenesis and the pathway for targeting proteins to the rhoptries have not been elucidated. Using the maturation of the Rhoptry-Associated Protein 1 (RAP-1) gene product to study these steps, it is reported here that a secretory pathway controls transport of protein complexes containing RAP-1 products to the rhoptries. Both brefeldin A (BFA) and low temperature reversibly block the processing of an 86-kDa precursor (Pr86) to the mature 82-kDa RAP-1 product (p82). Furthermore, the points of action of BFA and low temperature appear to overlap since their sequential application reversibly prevents Pr86 processing. Treatment of intact cells with N-ethylmaleimide, which prevents the fusion of transport vesicles with Golgi membranes in other eukaryotic cells, irreversibly blocks processing of Pr86. The role of the secretory pathway in targeting p82 protein complexes to the rhoptries product of RAP-1. These in vitro results also reveal that the RAP-1 product contains a cleavable N-terminal signal peptide and appears to be initially synthesized as an 84-kDa protein. The above data indicate that transport of p82 to the rhoptries is regulated by the secretory pathway and that the RAP-1 primary translation product differs in apparent molecular weight from the in vivo precursor Pr86. Our results suggest that rhoptry biogenesis is controlled in part by the secretory pathway and that the RAP-1 gene product acquires a previously undetected protein modification during its maturation.