AtT-20 cells contain two molecular weight forms (87 and 66 kDa) of the prohormone convertase PC1 (also known as PC3), thought to be involved in prohormone maturation. In this study we found that PC1 is first synthesized as a 94-kDa protein, which is then rapidly converted to a 84-kDa form. Two lines of evidence suggest that the generation of the 84-kDa protein from its 94-kDa precursor occurs in the endoplasmic reticulum (ER). The processing of the 94-kDa protein to the lower molecular weight form was extremely rapid, occurring with a half-life less than 2 min. The 84-kDa form was initially endoglycosidase H-sensitive, indicating lack of acquisition of sugars transferred in the medial Golgi. Within 40 min after the labeling period, the 84-kDa protein was converted to an endoglycosidase H-resistant form of 87 kDa, which was then processed to an endoglycosidase H-resistant 66-kDa protein. Radiosequencing of the 87- and 66-kDa proteins indicated that the biosynthesis of the 87-kDa protein involves the removal of the 83 amino acid Pro segment and that the processing of the 87-kDa to the 66-kDa form occurred by cleavage of a carboxy-terminal segment. Brefeldin A did not interrupt the cleavage of the 94-kDa to the 87-kDa protein, but completely blocked the processing of the 84- to 87-kDa proteins to the 66-kDa species. The 84-kDa protein produced in brefeldin-treated cells remained sensitive to endoglycosidase H, indicating a lack of exposure to Golgi sugar transferases.(ABSTRACT TRUNCATED AT 250 WORDS)