Recently, glycine-extended processing intermediates of progastrin were identified in porcine stomach using a radioimmunoassay with conventional polyclonal antisera developed against a synthetic peptide analogue for progastrin processing intermediates, gastrin 6-G(Tyr-Gly-Trp-Met-Asp-Phe-Gly). We developed monoclonal antibodies specific for glycine-extended processing intermediates of progastrin (gastrin G). Monoclonal antibody 109-21 appeared to require the carboxyl-terminal pentapeptide structure of gastrin 6-G for maximal binding. Cross-reactivities of 109-21 against gastrin 17 I, gastrin 17 II, cholecystokinin-octapeptide, des(SO3) cholecystokinin-octapeptide, and gastrin 6-G-R-R were respectively 1%, less than 0.1%, less than 0.1%, 0.1%, and 0.5%. With this monoclonal antibody and a polyclonal gastrin antibody we examined the concentrations of gastrin and gastrin G in tissue and the effects of bombesin on the release of gastrin and gastrin G from rat antral mucosa in tissue culture. The gastrin G to gastrin ratio was 2.2 in rat antral mucosa and 0.66 in rat duodenal mucosa. In tissue culture, bombesin significantly stimulated gastrin and gastrin-G secretion at doses of 10(-8) and 3 X 10(-8) M. Atropine (10(-6) M) abolished the actions of carbachol to stimulate gastrin and gastrin-G secretion but had no effect on bombesin-stimulated gastrin and gastrin-G secretion. These results suggest that gastrin G is cosecreted with gastrin in response to carbachol and bombesin, and the stimulation of gastrin and gastrin-G secretion by bombesin does not involve cholinergic neural pathways and may reflect a direct action on gastrin cells.