GATA-4 and GATA-6 are zinc finger transcription factors that regulate gene expression, differentiation, and cell proliferation in various tissues. These factors have been implicated in the development of endodermal derivatives, including epithelial cells in the yolk sac, lung, and stomach. In the present study, we have characterized the expression of GATA-4 and GATA-6 during development of another endodermal derivative, the mouse pancreas, using a combination of in situ hybridization and immunohistochemistry. Neither GATA-4 nor GATA-6 antigen was detected in E10.5 pancreatic epithelial buds expressing Pdx-1. By E15.5, GATA-4 mRNA and protein were evident in developing pancreatic acini, but not in ductal or endocrine cells of the pancreas; GATA-6 mRNA and protein were present in both endocrine and exocrine cell precursors. In the newborn and adult pancreas, GATA-4 protein was seen in acinar cells, while GATA-6 antigen was found mainly in islet beta-cells. The amphicrine pancreatic AR42J-B13 cell line was used to study the expression of GATA-4 and GATA-6 during the differentiation of these cells towards an endocrine phenotype. Endocrine differentiation was associated with marked increase in GATA-6 but not GATA-4 mRNA levels. We conclude that GATA-4 is a marker of exocrine pancreatic differentiation, whereas GATA-6 is a marker of endocrine pancreatic development.