A method is described for the isolation and short-term culture of canine antral gastrin (G) cells. Tissue was dispersed by enzymes and G cells enriched by elutriation and cultured for 40 h. These cultures contained 12% G cells and less than 2% somatostatin- or serotonin-containing cells. Bombesin (0.001-100 pM) potently stimulated gastrin release from cell cultures in a linear fashion over 2 h. The bombesin-specific monoclonal antibody 2A11 dose-dependently blocked bombesin stimulation. Somatostatin (0.001-1,000 nM) inhibited bombesin-stimulated gastrin release. Antibody to somatostatin (Mab S8) prevented the inhibition by exogenous somatostatin but did not alter bombesin-stimulated or basal gastrin release. The substance P (SP) analogue spantide (1 nM-1 microM) did not inhibit bombesin-stimulated gastrin release. Postreceptor activation of adenylate cyclase by forskolin and of protein kinase C by the phorbol ester, beta-TPA, caused gastrin release. The calcium ionophore A23187 also released gastrin in a dose-dependent fashion. This methodology allows enrichment and short-term culture of antral G cells; these cells have stimulatory bombesin and inhibitory somatostatin receptors, suggesting that these peptides have a direct action on antral G cells. Furthermore, G cells are activated by cAMP and calcium/phosphatidylinositol-dependent mechanisms.