The nature of the primary genetic defects in ob/ob and db/db mice are unknown. Both the obese (ob) and diabetes (db) mutations produce similar, multicomponent obese-hyperinsulinemic syndromes when maintained in the same strain of mouse. In an attempt to find differences between these mutations in neuroendocrine function affecting the islets of Langerhans or the pituitary, tissue content of four neuropeptides that are known to be capable of influencing the rate of insulin secretion was examined in obese (ob/ob) and diabetes (db/db) mice. In the first study, C57BL/6J ob/ob and control males were studied at 3, 4, and 11 weeks of age. In the second study, db/db mice of both sexes and two inbred strains (C57BL/6J and C57BL/KsJ), which differ markedly in the severity of expression of the diabetes phenotype, were studied at 3 weeks of age, before the development of hyperglycemia and secondary consequences thereof. Immunoreactive peptides were measured in acetic acid extracts of pancreas and pituitary. No differences between male ob/ob and db/db mice of the C57BL/6J strain were found. Marked sex differences in lean control mice were found at 3 weeks of age in pancreatic Met-enkephalin-LI and galanin-LI (with two- to threefold higher content in males). Low pancreatic content (50% to 70% lower than in control mice) of galanin-LI, Met-enkephalin-LI and Leu-enkephalin-LI was associated with hyperinsulinemia in male B6 ob/ob and db/db mice at 3 weeks of age, though not in B6 db/db females and not in BKs db/db mice of either sex. The marked strain and sex differences in pancreatic neuropeptide content in control mice of both B6 and BKs strains, which were demonstrated in very young, unweaned mice, suggest that somewhat different metabolic control mechanisms may operate in the two sexes. These observations may be related to the strain and sex differences in penetrance or severity of the diabetes syndrome that have been previously described.