The precursor of the acid-stimulating hormone gastrin gives rise to multiple peptides differing markedly in biological activity, but the relevant biosynthetic pathways are poorly understood. We have used antibodies to amidated gastrins, gastrins with COOH-terminal glycine (Gly) gastrins with COOH-terminal hydroxyglycine (GlyOH) and to the COOH terminus of progastrin, to immunoprecipitate peptides labeled with [35S]sulfate or [3H]tyrosine during incubation of rat antral mucosa in vitro. Labeled progastrin was detectable after 30 min of continuous incubation with isotopic precursors, G34 and G34-Gly after 60 min, and G17 and G17-Gly after 120 min. Pulse chase experiments indicated that progastrin is converted to G34-Gly which then follows one of two pathways: (a) hydroxylation of COOH-terminal Gly and conversion to G34 followed by cleavage yielding G17, or (b) cleavage to G17-Gly. The kinetics of G17-Gly and G17 labeling were similar, suggesting that G17-Gly is a product in its own right, and not simply an intermediate in G17 synthesis. Since the two peptides are reported to have distinct biological activities, they appear to be alternative mature products of progastrin processing.