Gene inactivation studies have shown that members of the Gata family of transcription factors are critical for endoderm development throughout evolution. We show here that Gata-4 and/or Gata-6 are not only expressed in the adult exocrine pancreas but also in glucagonoma and insulinoma cell lines, whereas Gata-5 is restricted to the exocrine pancreas. During pancreas development, Gata-4 is expressed already at embryonic d 10.5 and colocalizes with early glucagon+ cells at embryonic d 12.5. Gata-4 was able to transactivate the glucagon gene both in heterologous BHK-21 (nonislet Syrian baby hamster kidney) and in glucagon-producing InR1G9 cells. Using gel-mobility shift assays, we identified a complex formed with nuclear extracts from InR1G9 cells on the G5 control element (−140 to −169) of the glucagon gene promoter as Gata-4. Mutation of the GATA binding site on G5 abrogated the transcriptional activation mediated by Gata-4 and reduced basal glucagon gene promoter activity in glucagon-producing cells by 55%. Furthermore, Gata-4 acted more than additively with Forkhead box A (hepatic nuclear factor-3) to trans-activate the glucagon gene promoter. We conclude that, besides its role in endoderm differentiation, Gata-4 might be implicated in the regulation of glucagon gene expression in the fetal pancreas and that Gata activity itself may be modulated by interactions with different cofactors.