Pancreatic-duodenal homoeobox factor-1 (PDX1) is a homoeodomain transcription factor that plays an important role in linking glucose metabolism in pancreatic β cells to the regulation of insulin gene transcription. Our previous results indicated that glucose activates PDX1 DNA-binding activity and insulin promoter activity via a stress-activated signalling pathway involving phosphatidylinositol 3-kinase (PtdIns 3-kinase) and stress-activated protein kinase 2 (SAPK2/p38). The present study was undertaken to determine the effects of other metabolizable and non-metabolizable nutrients. The results indicate that non-metabolizable nutrients, with the exception of 2-deoxyglucose, had no effect. Metabolizable nutrients that could stimulate calcium uptake and insulin release were shown to activate both PDX1 and the insulin promoter. The possible role of insulin acting via an autoregulatory loop was therefore examined. Insulin was shown to potently activate PDX1 DNA-binding activity and insulin promoter activity. The effects of insulin were inhibited by the PtdIns 3-kinase inhibitors wortmannin and LY294002 and by the SAPK2 inhibitor SB203580, suggesting that its effects were mediated via activation of PtdIns 3-kinase and SAPK2. Further support for the insulin-mediated activation of SAPK2 came from the observation that both glucose and insulin stimulated the phosphorylation of SAPK2. These results suggest that both glucose and insulin stimulate PDX1 DNA-binding activity and insulin promoter activity via a pathway involving PtdIns 3-kinase and SAPK2.