Receptor-dependent and -independent regulation of gastrin secretion from cultured human antral G cells was investigated. Human antral mucosal cell preparations that were enriched for G cells were obtained by sequential incubations with collagenase and ethylenediaminetetraacetic acid, centrifugal elutriation, and short-term culture. After a 2-day incubation period, gastrin- and somatostatin-containing cells accounted for 15% and 5%, respectively, of the total adhered-cell population. Forskolin, A23187, and beta-phorbol 12 myristate 13-acetate stimulated basal gastrin secretion from cultured human G cells in a concentration-dependent fashion. These results indicate that gastrin release could be mediated by elevations in cytosolic cyclic adenosine monophosphate levels, calcium influx, or activation of protein kinase C. A direct stimulatory role for bombesin- and gastrin-releasing peptide was supported by experiments showing concentration-dependent enhancement of gastrin release by bombesin from 0.01 fmol/L to 10 nmol/L. The putative bombesin antagonist [Leu13-psi-CH2NH-Leu14] bombesin augmented basal gastrin levels by itself and produced weak inhibition of bombesin-induced gastrin secretion from human antral G cells. Somatostatin potently suppressed forskolin- and bombesin-mediated gastrin release but did not significantly alter basal gastrin levels. These results suggest that bombesin and somatostatin directly activate and inhibit G-cell function via specific and sensitive receptors. Furthermore, the adenylate cyclase and phosphatidyl inositide second messenger systems seem to be intracellular mediators of gastrin secretion from human antral G cells.