The major hydrolytic degradation pathways of glucagon under acidic conditions are cleavage at Asp-9, Asp-15, and Asp-21, and deamidation at Gln-3, Gln-20, Gln-24, and Asn-28. The rate constants for these pathways were determined in the pH range 1-2.4 at 60 o C by kinetic data analysis of substrate and degradation product concentration-time profiles. Deamidation kinetics were determined using penta-peptide fragments of glucagon containing the labile amide residue. The accurate determination of the cleavage rate constants was confounded by the complexity of the degradation scheme of glucagon. Peptide cleavage kinetics were determined by degradation of glucagon and its cleavage fragments under identical conditions and the use of area-under-the-curve (AUC) and nonlinear regression methods of analysis. Glucagon degradation was first-order with respect to time and concentration in the range of 31-00μM. Glutaminyl deamidation rate constants were first-order with respect to hydronium ion concentration and were similar for all three residues indicating a lack of sequence effects. The rate constants for Asp cleavage were not first-order with respect to hydronium ion concentration and cleavage at Asp-21 was slower than cleavage at Asp-9 and Asp-15 over the studied pH range.