Insulin secretory physiology has been characterized in tumor cell lines derived by primary culture of insulinomas that developed in transgenic mice expressing the large T-antigen of SV40 in pancreatic islet β-cells. Cells in one of these lines, βTC-3, contain large amounts of insulin (3100 ± 294 ng/100 μg cellular protein). Constitutive release of insulin over 2 h in static incubation was low at 31.9 ng/100 μg protein and was increased 2-fold by glucose (16.7 mM) and 8-fold by depolarizing concentrations of potassium (45 mM). Isobutylmethylxanthine (IBMX; 0.5 mM) and forskolin (5 and 50 μM), which elevated cellular levels of cAMP, were ineffective as secretagogues, but dramatically potentiated glucose and potassium effects on insulin release (6.5-and 4-fold, respectively). A variety of other known insulin secretagogues stimulated insulin release in a manner analogous to their effects in normal islets. The sulfonylurea glipizide (1 μM) and the tumor-promoting phorbol ester 12-O-tetradeca-noylphorbol-13-acetate (1 μM) stimulated insulin release 3.4-and 13.7-fold, respectively. The cholinergic agonist carbachol (2 μM) was ineffective alone, but potentiated glucose-induced insulin release 2.8-fold. Comparable stimulation of insulin release by glucose (16.7 mM) and glucose (16.7 mM) plus IBMX (0.5 mM) was noted with several other βTC lines, which were derived independently from separate transgenic miceGlucose-and glucose-plus IBMX (0.5 mM)-induced insulin release occurred progressively from 0.15-16.7 mM, indicating that insulin release from βTC-3 cells occurred at much lower levels than that from normal islets. However, as in the normal islet, the glucose concentration dependency for insulin release was highly correlated (r = 0.93) with the glucose concentration dependency for glucose utilization (measured by3H20 formation from [53H]glucose). This suggests that glucose induces insulin release from βTC-3 cells by a mechanism similar to that in the normal isletThe high insulin content, the multifold stimulation of insulin release by a variety of secretagogues, their convenient propaga-tion in culture, and the renewable source of these cell lines make the βTC cells a convenient model for studies of β-cell function.