Sucrose- and fructose-enriched diets produce hepatic insulin resistance in rats independently of obesity. In humans, fructose infusion results in impaired insulin regulation of glucose production. The aim of the present study was to identify intrahepatic mediators of sucrose- and fructose-induced hepatic insulin resistance. In study 1, male rats were fed a control diet (STD, 68% of energy from corn starch, 12% from corn oil) or a sucrose-enriched diet (HSD, 68% sucrose, 12% corn oil) for 1, 2, or 5 wks. HSD produced hepatic insulin resistance at all time points. Hepatic protein tyrosine phosphatase 1B protein levels and activity were increased at 5 wks only, whereas c-jun N-terminal kinase (JNK) activity was increased at all time points. Normalization of JNK activity in hepatocytes isolated from HSD rats improved insulin-stimulated tyrosine phosphorylation of IRS proteins and insulin suppression of glucose release. In study 2, male rats were provided STD for 1 wk and then were either fasted or fasted and refed either STD or HSD for 3h or 6h. Rats refed HSD were characterized by increased hepatic JNK activity and phosphorylation of IRS1 on serine 307 after 6h only. In study 3, hyperglycemic, hyperinsulinemic pancreatic clamps were performed for 3 or 6h in the presence or absence of low or high intraportal fructose infusions. High intraportal fructose infusions, which increased portal vein fructose concentration to ~ 1mM, increased hepatic JNK activity and phosphorylation of IRS1 on serine 307 at 6h only. These data suggest that sucrose- and fructose-induced hepatic insulin resistance are mediated, in part, via activation of JNK activity. Thus, high rates of fructose metabolism in the liver appear to acutely activate stress pathways.