Plasma glucose, immunoreactive glucagon (IRG), and insulin were measured in hypophysectomized dogs receiving cortisol and thyroid replacement therapy. 4 wk after hypophysectomy mean fasting plasma glucose levels had declined from 90±2 mg/100 ml to 64±2; fasting and arginine-stimulated insulin and IRG levels were, respectively, ∼50% lower and unchanged. 12 wk or more after hypophysectomy, despite lower plasma glucose levels, fasting and arginine-stimulated IRG levels were significantly below control dogs. Hypophysectomized and shamhypophysectomized dogs were subjected to total pancreatectomy. Postoperatively, in the sham-hypophysectomized, depancreatized dogs fasting glucose levels ranged from 300-500 mg/100 ml on 8-10 U/day of insulin; IRG levels averaged 215±29 pg/ml. The hypophysectomized, depancreatized dogs required 0-4 U/day and fasting glucose levels under 100 mg/100 ml were not uncommon, even without insulin; fasting IRG levels averaged 63±4 pg/ml (P < 0.001). During arginine infusion in sham-hypophysectomized, depancreatized dogs, IRG levels rose from 215±60 pg/ml to a peak of 404±112 pg/ml; in hypophysectomized, depancreatized dogs, the base line IRG averaged 44±8 and the peak 110±25 pg/ml (P < 0.05). IRG levels in the venous effluent of the gastric fundus, the major source of nonpancreatic glucagon, reached a peak of 4,898±959 pg/ml in the sham-hypophysectomized, depancreatized group during arginine infusion and only 219±128 pg/ml in the hypophysectomized, depancreatized group. In three hypophysectomized, depancreatized dogs, a replacement infusion with glucagon for 10 h promptly increased hyperglycemia by 80-180 mg/100 ml and worsened glycosuria, evidence of a hepatic response to glucagon replacement. It is concluded that hypophysectomy somehow decreased both the hypersecretion of gastric IRG and the severe hyperglycemia that otherwise follows pancreatectomy. The hypophysectomized, depancreatized animal, therefore, has combined insulin and glucagon deficiency, and the latter may contribute to reduced severity of its hyperglycemia.