Serum glucose and plasma C-peptide response to IV glucagon administration was evaluated in 24 healthy dogs, 12 dogs with untreated diabetes mellitus, 30 dogs with insulin-treated diabetes mellitus, and 8 dogs with naturally acquired hyperadrenocorticism. Serum insulin response also was evaluated in all dogs, except 20 insulin-treated diabetic dogs. Blood samples for serum glucose, serum insulin, and plasma C-peptide determinations were collected immediately before and 5,10,20,30, and (for healthy dogs) 60 minutes after IV administration of 1 mg glucagon per dog. In healthy dogs, the patterns of glucagon-stimulated changes in plasma C-peptide and serum insulin concentrations were identical, with single peaks in plasma C-peptide and serum insulin concentrations observed approximately 15 minutes after IV glucagon administration. Mean plasma C-peptide and serum insulin concentrations in untreated diabetic dogs, and mean plasma C-peptide concentration in insulin-treated diabetic dogs did not increase significantly after IV glucagon administration. The validity of serum insulin concentration results was questionable in 10 insulin-treated diabetic dogs, possibly because of anti-insulin antibody interference with the insulin radioimmunoassay. Plasma C-peptide and serum insulin concentrations were significantly increased (P < .001) at all blood sarnplkg times after glucagon administration in dogs with hyperadrenocorticism, compared with healthy dogs, and untreated and insulin-treated diabetic dogs. Five-minute C-peptide increment, C-peptide peak response, total C-peptide secretion, and, for untreated diabetic dogs, insulin peak response and total insulin secretion were significantly lower (P < .001) in diabetic dogs, compared with healthy dogs, whereas these same parameters were significantly increased (P < .011 in dogs with hyperadrenocorticism, compared with healthy dogs, and untreated and insulin-treated diabetic dogs. Although not statistically significant, there was a trend for higher plasma C-peptide concentrations in untreated diabetic dogs compared with insulin-treated diabetic dogs during the glucagon stimulation test. Baseline C-peptide concentrations also were significantly higher (P < .05) in diabetic dogs treated with insulin for less than 6 months, compared with diabetic dogs treated for longer than 1 year. Finally, 7 of 42 diabetic dogs had baseline plasma C-peptide concentrations greater than 2 SD (ie, >0.29 pmol/mL) above the normal mean plasma C-peptide concentration; values that were significantly higher, compared with results in healthy dogs (P < .001) and with the other 35 diabetic dogs (P < .001). In summary, measurement of plasma C-peptide concentration during glucagon stimulation testing allowed differentiation among healthy dogs, dogs with impaired β-cell function (ie, diabetes mellitusl, and dogs with increased β-cell responsiveness to glucagon (ie, insulin resistance). Plasma C-peptide concentrations during glucagon stimulation testing were variable in diabetic dogs and may represent dogs with type-1 and type-2 diabetes or, more likely, differences in severity of β-cell loss in dogs with type-1 diabetes. J Vet Intern Med 1996;10:116–122. Copyright © 1996 by the American College of Veterinary Internal Medicine.