The glycation of immunoreactive insulin (IRI) was assessed in extracts of pancreas and islets from control and hyperglycemic animal models. Glycated and nonglycated IRI were separated by affinity chromatography and quantified by radioimmunoassay. Hydrocortisone-treated Wistar rats (80 mg · kg⁻¹ · day⁻¹ and obese hyperglycemic (ob/ob) mice showed significant increases in plasma glucose (P < 0.001), percentage glycated hemoglobin (P < 0.001), plasma IRI (P < 0.01), and total pancreatic IRI content (P < 0.01), compared with their respective controls. These diabetic groups also demonstrated significant increases (P < 0.05) in the percentage of glycated pancreatic IRI above the controls. Streptozotocin-treated (200 mg/kg) Swiss TO mice exhibited significant increases in plasma glucose (P < 0.001), glycated hemoglobin (P < 0.001), and percentage glycated pancreatic IRI (P < 0.05), compared with untreated controls, despite a marked decrease in both plasma IRI (P < 0.001) and total pancreatic IRI content (P < 0.001). Significant elevations in the percentage of glycated IRI were also observed in islets isolated from obese hyperglycemic (ob/ob) mice (P < 0.001), compared with islets from lean controls, and when lean mouse islets were cultured in hyperglycemic media for 24 h (33.3 vs. 5.6 mmol/l D-glucose; P < 0.001). The contribution of glycated plus nonglycated insulin and proinsulin to the total IRI was estimated in lean and obese mouse pancreatic extracts following high-performance liquid chromatography separation. The contribution of proinsulin to the total IRI was ∼ 10%. Proinsulin represented 27–28% of the total glycated IRI. These data indicate that the glycation of insulin and proinsulin occurs within the pancreatic islets and is elevated in both insulin-deficient and insulin-resistant diabetic animal models.