Patients with noninsulin-dependent diabetes (NIDDM) show an increase in the relative plasma levels of proinsulin and proinsulin conversion intermediates, which is corrected by strict glycemic control. This observation suggests that hyperglycemia per se may be responsible for generating the aberrant plasma hormone profile. The question remains, however, whether a genetic predisposition to NIDDM underlies the failure of the insulin production machinery to meet a prolonged increase in secretory demand. In this study, islet monolayer cultures from the diabetes-prone Psammomys obesus and normal diabetes-resistant rats were exposed to RPMI 1640 medium containing either 11.1 or 33.3 mM glucose; insulin-related peptides were resolved by HPLC. Prolonged exposure (10 days) of rat islets to high glucose resulted in a reduced a secretory response to an acute glucose stimulus associated with a 37% reduction in the insulin content but no change in the proinsulin/insulin ratio. When subjected to a similar protocol, islets from prediabetic Psammomys lost the insulin response to glucose; β-cell insulin content was reduced by about 70%, and the proportion of proinsulin- related peptides increased from 18% to 38%. In the in vivo situation pancreatic extracts from nonfasted diabetic Psammomys contained 36% proinsulin-related peptides in contrast to 15% in pancreatic extracts from nondiabetic animals. Thus, prolonged in vitro exposure of prediabetic Psammomys islets to high levels of glucose could reproduce could reproduce the modified β-cell secretory profiles observed in vivo in the diabetic animal. These results support the hypothesis that hyperproinsulinemia in NIDDM is secondary to the inability of β-cells to meet a sustained increase in insulin demand, whereas individuals with normal β-cells may meet such demand with an adequate output of mature insulin.