We investigated the specific and associated effects of insulin and glucose on β-cell growth and function in adult rats. By combining simultaneous infusion either of glucose and/or insulin or glucose and diazoxide, three groups of rats were constituted: hyperglycemic-hyperinsulinemic rats (high glucose–high insulin), hyperglycemic-euinsulinemic rats (high glucose), and euglycemic-hyperinsulinemic rats (high insulin). All the infusions lasted 48 h. Control rats were infused with 0.9% NaCl (saline controls). In all groups, β-cell mass was significantly increased, compared with controls (by 70% in high glucose–high insulin rats, 65% in high glucose rats, and 50% in high insulin rats). The stimulation of neogenesis was suggested by the high number of islets budding from pancreatic ducts in high glucose–high insulin and high glucose rats and by the presence of numerous clusters of few β-cells within the exocrine pancreas in high insulin rats. β-Cell hypertrophy was observed only in high glucose–high insulin rats. The rate of β-cell proliferation was similar to that of controls in high glucose–high insulin rats after a 48-h glucose infusion, dropped dramatically in high insulin rats, and dropped to a lesser extent in high glucose rats. In high glucose–high insulin and high glucose rats, β-cell mass increase was related to a higher β-cell responsiveness to glucose in vitro as measured by islet perifusion studies, whereas in high insulin rats, no significant enhancement of glucose induced insulin secretion could be noticed. The data show that glucose and insulin may have specific stimulating effects on β-cell growth and function in vivo in adult rats independently of the influence they exert each other on their respective plasma concentration.