To determine if the loss of the glucagon response to glucopenia that follows destruction of beta cells is at least in part a consequence of the absence of the normal glucopenia-induced decline in insulin secretion, pancreata from insulin-requiring streptozotocin-diabetic rats were studied. In the absence of insulin, a reduction in perfusate glucose concentration from 150 to 25 mg/dl failed to elicit a rise in glucagon concentration. When insulin was co-perfused at 30 mU/ml, the estimated within-islet concentration of insulin under these circumstances, but discontinued during the glucopenic interval, reducing the insulin concentration in the pancreatic venous effluent from ∼26 mU/ml to <100 μU/ml, a prompt and significant rise in glucagon was observed until glucose and insulin levels were raised to their original concentrations. The rise in glucagon, which was approximately 25% of the normal response, did not occur when insulin concentration in the perfusate was maintained at 30 U/ml during the glucopenic period. Nor did it occur when insulin was perfused at 360 μU/ml and discontinued during the glucopenic period, thereby lowering insulin in the venous effluent from 300 μU/ml to 5 μU/ml. It is concluded that the decline in insulin from its normal concentrations within the islets makes a modest but significant contribution to the rise in glucagon that occurs during glucopenia.