Immunoreactive TRH (IR-TRH) has been found in the mammalian pancreas, with several studies documenting high concentrations in the late fetal/early neonatal period. As the factors regulating pancreatic TRH synthesis and release have not been fully explored, we developed a monolayer culture system of dissociated fetal/neonatal rat pancreatic cells to study the release of TRH from the mammalian pancreas. IR-TRH was detected in the culture medium and the IR material appeared authentic based on parallelism with synthetic TRH in RIA and retention time on HPLC. Potassium-induced depolarization (60 mM KCl) resulted in a 170% increase in TRH release compared to that by the Krebs-Ringer bicarbonate control (P less than 0.05). Serotonin stimulated TRH release, with the maximal effect seen with 10(-6) M (130% increase compared to control; P less than 0.05). Carbachol resulted in a dose-dependent inhibition of TRH release (57% inhibition of release at 10(-8) M; P less than 0.01 compared to control). There was no effect on release with norepinephrine, epinephrine, dopamine, gamma-aminobutyric acid, or histamine. We conclude the following. 1) Authentic TRH is secreted by fetal/neonatal rat pancreatic cells in culture. 2) The secretion of TRH is stimulated by potassium-induced depolarization in a calcium-dependent manner, suggesting a classic neurosecretory process of release. 3) The secretion of pancreatic TRH may be under specific neurotransmitter control, with serotonin stimulating and acetylcholine inhibiting release of the tripeptide.