Abstract Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg 94Arg 95 and Lys 74Lys 75 residues generating gastrin heptadecapeptide (G17-NH 2). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH 2) with little processing at Lys 74Lys 75. Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH 2. To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys 74Lys 75) or mutant (Arg 74Arg 75, Lys 74Arg 75, and Arg 74Lys 75) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH 2 formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys 74Lys 75 site within G34-NH 2 to Lys 74Arg 75 resulted in the production of primarily G17-NH 2 rather than G34-NH 2, inhibition of PC1/PC3 did not significantly inhibit processing at the Lys 74Arg 75 site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells.