The actions of transforming growth factor-β isoforms as potent regulators of growth and differentiation have led to the examination of their presence in the human pancreas. The cellular localization of TGF-β1, TGF-β2, and TGF-β3 was assessed in the normal human pancreas by using immunohistochemical and in situ hybridization techniques. Although cytoplasmic immunoreactivity for TGF-β1, TGF-β2, and TGF-β3 was found in islet cells, acinar cells, and ductal cells, a differential immunostaining pattern for TGF-β isoforms was observed. In the endocrine pancreas, the islet cells demonstrated diffuse cytoplasmic immunostaining for TGF-β1, TGF-β2, and TGF-β3. However, only TGF-β2 and TGF-β3 exhibited an intense pattern of immunostaining in a few endocrine cells. Most of the positive islet cells coexpressed insulin. In contrast, in the exocrine pancreas, a greater number of acinar cells showed immunoreactivity for TGF-β1 than for TGF-β2 and TGF-β3. In the ductal cells, all three TGF-β isoforms showed a similar intensity and pattern of immunostaining and were observed more frequently in the smaller distal ductules than in the larger pancreatic ducts. TGF-β1 and TGF-β3, but not TGF-β2, immunostaining was detected strongly in the smooth muscle cells and weakly in the endothelial cells of the blood vessels, whereas the fibroblasts of the interstitium were completely negative. In situ hybridization revealed that mRNA encoding all three TGF-β isoforms colocalized with their respective proteins in islets, acinar cells, and ductal cells. In contrast, mRNA expression was absent in the smooth muscle cells and endothelium of the vessels. These results suggest that TGF-β isoforms may act by both autocrine and paracrine mechanisms in the pancreas. The differential pattern of expression observed for each TGF-β isoform implies unique roles for these proteins in the regulation of the endocrine and exocrine pancreas.