1. Hyperinsulinaemia has been reported to be an independent risk factor for cardiovascular diseases. Insulin stimulates both the phosphatidylinositol 3-kinase (PI3-K)/Akt and mitogen-activated protein kinase (MAPK) pathways. To investigate the direct effects of insulin on vascular tissues, we examined the gene and protein expression of insulin signalling molecules, endothelial nitric oxide synthase (eNOS) and MAPK in aortas obtained from established hyperinsulinaemic rats under deep urethane anaesthesia (1.2 g/kg, i.p.). 2. High plasma insulin levels significantly enhanced the gene and protein expression of eNOS in aortas. This was accompanied not only by increased mRNA levels of insulin receptor substrate (IRS)-1, IRS-2, PI3-K and Akt, but also by a high protein content of Akt and phospho-Akt (Ser473). 3. In contrast, MAPK mRNA levels were decreased in hyperinsulinaemic rats compared with normoinsulinaemic rats. 4. Insulin receptor mRNA levels were also lower in insulin-treated rats rather than controls. The overexpression of mRNA for vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-I receptor was also observed in aortas from hyperinsulinaemic rats. 5. To our knowledge, these data provide the first direct measurements of the mRNA of insulin signalling molecules and the downstream eNOS and MAPK. We conclude that hyperinsulinaemia itself can lead to the upregulation of eNOS and the PI3-K/Akt pathway in the vasculature and may also induce the overexpression of VEGF and IGF-I receptor genes.