The insulin promoter is both positively and negatively regulated in response to conditions to which pancreatic beta-cells are exposed. Exposure of intact rat islets and INS-1 pancreatic beta-cells to 11 mm glucose for minutes to hours results in an enhancement in the rate of insulin gene transcription assessed with a reporter linked to the insulin gene promoter. In contrast, chronic exposure of rat islets or beta-cells to 11 mm glucose results in loss of the glucose responsiveness of the insulin gene promoter. By 48 h, glucose inhibits insulin gene promoter activity. Here we show that not only the acute effect of elevated glucose to stimulate the insulin gene promoter but also the chronic effect of elevated glucose to inhibit the insulin gene promoter depend on ERK1/2 mitogen-activated protein kinase activity. In examining the underlying mechanism, we found that acute exposure to 11 mm glucose resulted in the binding of the transcription factors NFAT and Maf to the glucose-responsive A2C1 element of the insulin gene promoter. An NFAT and C/EBP-beta complex was observed in cells chronically exposed to 11 mm glucose. Formation of NFAT-Maf and NFAT-C/EBP-beta complexes was sensitive to inhibitors of ERK1/2 and calcineurin, consistent with our previous finding that activation of ERK1/2 by glucose required calcineurin activity and the well documented regulation of NFAT by calcineurin. These results indicate that the ERK1/2 pathway modulates partners of NFAT, which may either stimulate or repress insulin gene transcription during stimulatory and chronic exposure to elevated glucose.