Some of the mechanisms underlying intestinal glucagon-like immunoreactive (GLI) peptide secretion from cultured fetal rat intestinal cells were investigated using modulators of the adenylate cyclase pathway [(Bu)2cAMP, theophylline, isobutylmethylxanthine], calcium fluxes (ionomycin, A23187), and protein kinase-C (phorbol ester). All of these agents were found to stimulate GLI peptide release, to 120-230% of paired control values (P less than 0.05-0.001). (Bu)2cAMP, but not the phorbol ester, also increased the total cell content of GLI peptides over the 2-h incubation period (P less than 0.05). No synergism between any of the three pathways was detected. When the mol wt distribution of the stored and secreted GLI peptides was determined in control and (Bu)2 cAMP-stimulated samples, 68 +/- 2% of the peptide corresponded to glicentin, while the remainder eluted with the same distribution coefficient as oxyntomodulin. No 3.5K glucagon was detected in any of the extracts. GLI peptide secretion by the cells was not altered by several pancreatic glucagon secretagogues (cortisol, bombesin, and prostaglandins E1 and D2), but was stimulated by the opioid peptide beta-endorphin (1 microM; P less than 0.02). These studies have indicated that the control of secretion of fetal rat intestinal GLI peptides is complex, involving activation of any one or a combination of the three major second messenger systems. A role for the adenylate cyclase pathway in regulating GLI peptide biosynthesis is also suggested.