Gastrin requires extensive post-translational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine -amidating monooxygenase (PAM) resulting in carboxyl-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed ³⁵S-Met-labeled pulse-chase biosynthetic experiments in canine antral G-cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G-cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor, ascorbic acid. To determine if one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G-cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabelled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G-cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.