Pancreatic islets from obese hyperglycemic mice are large and contain a high proportion of normally functioning beta-cells. We have previously shown that young obese mice have an elevated beta-cell proliferation rate at 3 weeks of age. We now wanted to investigate possible factors involved in the initiation of islet growth, including blood glucose, C peptide, glucagon-like peptide-1, vasoactive intestinal polypeptide, and L-5-hydroxytryptophan. We found that the increased beta-cell proliferation on day 20 precedes the rise in blood glucose by 2 days. The islet cell proliferation, measured as the 5-bromo-2'-deoxyuridine labeling index, in 20-day-old lean mice, was enhanced in a dose-dependent manner when glucagon-like peptide-1 or C peptide was injected s.c. for 2 days. L-5-Hydroxytryptophan inhibited the proliferation. C Peptide also increased the islet cell labeling index during islet culture. We conclude that in addition to the effect of glucose, islet proliferation can be triggered by other factors involved in the physiological regulation of increased insulin release. Stimulation of islet proliferation may be related to the actual release of insulin, and C peptide may function as a mediator of such responses.