Using laser-scanning confocal microscopy, we have monitored glucose-induced changes in the subcellular localization of insulin promoter factor-1 (IPF-1) labeled with a c-myc epitope tag. This construct trans-activated the insulin promoter in single living MIN6-beta-cells as assessed by luciferase-based promoter analysis. IPF-1.c-myc expression also enhanced the response of the insulin promoter to elevations in extracellular glucose concentration. In the majority (148/235, 63%) of cells maintained at low (3 mM) extracellular glucose concentration, IPF-1.c-myc immunoreactivity was confined to the nuclear periphery. Incubation of cells at stimulatory (30 mM) glucose concentrations caused a rapid redistribution of the chimera to the nucleoplasm (775/958, 81% of cells). By contrast, the irrelevant transcription factor c-Fos, tagged with either c-myc or as a chimera with luciferase, was localized exclusively to the nucleoplasm irrespective of the glucose concentration. Furthermore, IPF-1 extended with the bulky (27 kDa) enhanced green fluorescent protein (EGFP) group was confined largely to the nucleoplasm at all glucose concentrations tested and did not support trans-activation of the insulin promoter by glucose. Movement of endogenous IPF-1 from the nuclear periphery to the nucleoplasm may therefore increase the trans-activational capacity of this factor in native beta-cells exposed to high extracellular glucose concentrations.