Trefoil factors (TFFs) 1, 2, and 3 are expressed in mucosal epithelia. TFFs are particular abundant in the intestine in which they play a crucial role in maintenance and restitution of the epithelium. Because pancreas developmentally arises from the primitive foregut, we explored the expression of TFFs in the pancreas in man and rat. Immunocytochemical staining of adult human pancreas showed abundant TFF3 immunoreactivity in pancreatic islets and some duct cells, whereas weak TFF1 and no TFF2 staining were detected. In the islets TFF3 localized to most insulin and some glucagon and pancreatic polypeptide-producing cells. TFF3 immunoreactivity was colocalized with insulin and glucagon in distinct cell clusters in human fetal pancreas at wk 14 and in the newborn rat pancreas. In isolated human and rat islets, TFF3 and TFF1 mRNA was identified by RT-PCR, and TFF3 protein was detected in human pancreas and islets by ELISA. Exposure of neonatal rat islets or insulinoma cells to GH, a known beta-cell growth factor, resulted in markedly increased TFF3 but decreased TFF1 mRNA levels. The effect of GH on TFF3 expression was confirmed by Western blot. Culture of neonatal rat islets in the presence of TFF3 resulted in attachment and migration of the islet cells, but no effects on proliferation, insulin secretion or cytokine-induced apoptosis were seen. These data demonstrate expression of TFFs in the endocrine pancreas, but their possible functions remain unknown.