The release of glucagon from pancreatic and extrapancreatic sources was studied in normal rats and in rats carrying transplants of a MtT-W-15 tumor which secretes large quantities of growth hormone and prolactin. The tumor-bearing rats had high serum levels of A cell immunoreactive glucagon (IRGa), total immunoreactive glucagon (IRGT) and immunoreactive insulin (IRI) and an increased total glucagon and insulin content of the pancreas. Pancreatic islets isolated from tumor-bearing rats secreted more glucagon under basal conditions but did not respond significantly to low glucose stimulation. However, they contained more insulin per islet and secreted more insulin under basal and stimulated conditions. The serum IRGa response to arginine infusion in vivo was lower in the tumor-bearing than in the normal rats. The introduction of a 5% glucose solution into the small intestine caused similar increases in the level of serum IRGT in the two groups of rats. Thus, the tumor increased the total pancreatic glucagon content and basal secretion, blunted the A cell response to stimulation, but did not significantly alter the secretion of glucagon by the intestine. We attribute these responses to tumor-induced hypersomatotropinism although we cannot rule out an effect of the large amounts of circulating prolactin.