Currently there is intense interest to define the mechanism of action of glucagon-like peptide-1 (GLP-1) in regulating beta-cell function, including insulin gene transcription. In this study, GLP-1 (100 nmol/l), in the presence of glucose (11 mmol/l), induced a similar71-fold increase in insulin gene promoter activity in INS-1 pancreatic beta-cells, an effect that was an order of magnitude larger than with either stimulant alone. The response to GLP-1 was mimicked by forskolin and largely inhibited by the protein kinase A (PKA) inhibitors, H89 and myristoylated PKI(14--22) amide, indicating partial mediation via a cAMP/PKA pathway. Significantly, the actions of both GLP-1 and forskolin were abolished by the selective Ca(2+)/calmodulin-dependent phosphatase 2B (calcineurin) inhibitor, FK506, as well as by the chelation of intracellular Ca(2+) by BAPTA (bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate). Glucose and GLP-1 also synergistically activated NFAT (nuclear factor of activated T-cells)-mediated transcription from a minimal promoter construct containing tandem NFAT consensus sequences. Furthermore, two-point base pair mutations in any of the three identified NFAT sites within the rat insulin I promoter resulted in a significant reduction in the combined effect of glucose and GLP-1. These data suggest that the synergistic action of glucose and GLP-1 to promote insulin gene transcription is mediated through NFAT via PKA- and calcineurin-dependent pathways in pancreatic beta-cells.