Hyperglycaemia and diabetes are associated with enhanced oxidative stress, leading to the progression of diabetic vascular pathologies (Goldin et al., Circulation 2006;114: 597-605). The redox sensitive transcription factor Nrf2 mediates induction of protective genes, such as heme oxygenase-1 (HO-1), via activation of an antioxidant response element (ARE) (Mann et al., Cardiovasc. Res. 2007;75:261-274). HO-1 metabolises pro-oxidant heme to the antioxidants biliverdin and bilirubin (Siow et al. Redox Rep. 2007;12:11-15). The present study has examined the signalling pathways involved in high glucose enhanced superoxide generation, Nrf2 activation and HO-1 induction in bovine aortic endothelial cells (BAEC). Confluent monolayers were treated for 0-24 h with DMEM containing 25mM D-glucose, in the absence or presence of the superoxide (O2●-) scavengers SOD (200 Uml-1) and Tiron (10µM), inhibitors of NADPH oxidase (apocynin, 100 µM), flavoproteins (DPI, 10µM), eNOS (L-NAME, 50µM), or MAPK (SB203580, 10µM; SP600125, 20µM; U0126, 10µM). HO-1, nuclear Nrf2, and phosphorylated MAP kinases levels were determined by immunoblotting, Nrf2 translocation by immunofluorescence, and O2●- by L-012 chemiluminescence. Treatment of BAEC with 25mM D-glucose (0-24h), but not D-mannitol, elicited concentration- and time-dependent increases in O2●-, Nrf2 translocation and HO-1 expression. Increased HO-1 expression induced by high glucose was attenuated by Tiron and L-NAME, but not by extracellular SOD or NADPH oxidase inhibitors. Although high glucose induced rapid phosphorylation of p38MAPK and JNK, only inhibition of JNK abrogated high glucose-induced HO-1 expression, implicating the JNK signaling pathway in high glucose-induced activation of the Nrf2-ARE pathway and HO-1 expression.