The physiological regulation of intestinal proglucagon-derived peptide secretion has not been well studied. We have therefore used a fetal rat intestinal cell culture model to investigate the control of secretion of the gut glucagon-like immunoreactive (GLI) peptides by other intestinal regulatory peptides in vitro. Secretion of the intestinal GLI peptides was found to be stimulated in a dose-dependent fashion by the intestinal endocrine peptide, gastric inhibitory peptide (at ≥10^-10 M, P < 0.05), and by the neurocrine peptides, gastrin-releasing peptide (at ≥12 M, P < 0.05), and calcitonin gene-related peptide (at ≥10^-8 M, P < 0.05). Gastrin-releasing peptide and its amphibian equivalent, bombesin were equipotent in stimulating GLI peptide secretion. In contrast, the endocrine and neurocrine intestinal somatostatin-related peptides, somatostatin-28 and - 14, inhibited release of the GLI peptides, at concentrations of 10^-10 (P < 0.01) and 10^-8 (P < 0.01) M, respectively, with significant differences in potency between the two peptides detected at 10^-10 M (P < 0.05). The inhibitory effects of both somatostatin-28 and -14 could be blocked by preincubation of the cells with pertussis toxin (P < 0.05). Dose-dependent stimulation of gut GLI peptide secretion was also detected in response to treatment of cultured cells with sodium oleate (at 10^-4 M; P < 0.05), or with the cholinergic agonist bethanecol (at ≥ 100 nM; P < 0.05). Other endocrine [cholecystokinin, glucagon, glucagon-like peptide-l(l–37), glucagon-like peptide-l(7-37), glucagon-like peptide-2, neurotensin, and peptide YY] and neurocrine (vasoactive intestinal peptide) peptides, and the synthetic glucocorticoid, dexamethasone, were without effect on secretion of the gut GLI peptides, at doses of 10^-12 to 10^-6 M. The results of the present study therefore demonstrate that secretion of the intestinal proglucagon-derived peptides is under the regulatory control of a wide variety of intestinal endocrine and neurocrine peptides, as well as nutrients (fats) and neurotransmitters (acetylcholine). (Endocrinology128: 3175–3182, 1991)