A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of NEFA. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of β-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein (LP) diet and that rats show high NEFA levels, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/stress activated protein kinase 2 (SAPK2) protein expression, in islets from young rats fed low (6 %) or normal (17 %; control) protein diets and maintained for 48 h in culture medium containing 5·6 mmol/l glucose, with or without 0·6 mmol/l palmitic acid. We also measured glucose-induced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16·7 mmol/l glucose was reduced in LP compared with control rats. In the presence of NEFA, there was an increase in insulin secretion in both groups. At 2·8 mmol/l glucose, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16·7 mmol/l glucose, LP and control islets showed a severe reduction in glucose oxidation in the presence of NEFA. The PDX-1 and insulin mRNA were significantly higher when NEFA was added to the culture medium in both groups of islets. The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and control islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways.