RIN1046-38 cells (RIN-38) exhibit a passage-dependent reduction in both basal and glucose-regulated insulin secretion, accompanied by decreased insulin content. In an attempt to explain the mechanism of the gradual decrease in insulin production in cultured cells, we analyzed the insulin promoter activity and the levels of an important trans-activator of the insulin gene, PDX-1, as a function of aging in culture. We demonstrate that the decrease in insulin content and secretion is reflected in decreased promoter activity and is associated with a decrease in E47 and BETA2 nuclear factors, but with a paradoxical 3-fold increase in PDX-1 protein levels. To dissect the effect of increased PDX-1 from the decrease in the additional transcription factors on insulin promoter activity, we overexpressed PDX-1 protein in low passage RIN-38 cells by recombinant adenovirus technology. PDX-1 overexpression did not reduce E47 and BETA2 levels, but was sufficient to suppress rat insulin promoter activity in a dose-dependent manner. The fact that PDX-1 levels participate in trans-activation of insulin promoter activity was demonstrated in HIT-T15 cells. Treating HIT-T15 cells with 1-2 multiplicity of infection of AdCMV-PDX-1 increased rat insulin promoter activity, whereas higher doses repressed insulin promoter activity in these cells as in RIN-38 cells. Our data demonstrate that PDX-1 regulates transcription of the insulin gene in a dose-dependent manner. Depending on its nuclear dosage and the levels of additional cooperating transcription factors, PDX-1 may act as an activator or a repressor of insulin gene expression, such that low as well as high doses may be deleterious to insulin production.