Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with heterozygous deletion of the Chicken Ovalbumin Upstream Promoter- Transcription Factor II (COUP-TFII) gene or complete disruption of the Hepatocyte Nuclear Factor 4 alpha (HNF4alpha) gene in pancreatic beta cells have similar insulin secretion defects leading us to hypothesize that there is transcriptional cross-talk between these two nuclear receptors. Here we show specific HNF4alpha activation of a reporter plasmid containing the COUP-TFII promoter region in transfected pancreatic beta cells. A stable association of the endogenous HNF4alpha with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4alpha transactivation of COUP-TFII. Dominant negative suppression of HNF4alpha function decreased endogenous COUP-TFII expression and specific inactivation of COUP-TFII by short interfering (si)RNA caused HNF4alpha mRNA levels to decrease in 832/13 INS-1 cells. This positive regulation of HNF4alpha by COUP-TFII was confirmed by adenoviral overexpression of human (h)COUP-TFII which increased HNF4alpha mRNA in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore COUP-TFII could contribute to the control of insulin secretion through the complex HNF4alpha/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.