Nuclear factor κB (NFκB) plays a pivotal role in early gene responses by promoting messenger RNA (mRNA) synthesis for various cell-adhesion molecules and inducible nitric oxide synthase. In this study, we examined whether increases in glucose concentration enhance NFκB expression in nuclear fractions of endothelial cells by using electrophoretic mobility shift assay. Bovine aortic endothelial cells (BAECs) were incubated in media containing 5.5-35 mM glucose. NFκB activity was increased as early as 1 h (peak activation at 2-4 h) after incubation with 35 mM glucose compared with 5.5 mM. Similar increases at 2 h of incubation were observed by using 25 but not 15 mM glucose. Glucose-induced NFκB activation was blocked by inhibiting nuclear translocation by using a peptide (SN-50) containing the nuclear-localization sequence of NFκB p50 linked to a membrane-permeable motif of the sequence for Kaposi fibroblast growth factor. Co-incubation with a selective protein kinase C (PKC) inhibitor, calphostin C, produced a concentration-dependent inhibition of glucose-induced NFκB activation. Thus NFκB activation is an early event in response to elevations in glucose, which may elicit multiple pathways contributing to the origin of hyperglycemia-or diabetes-induced endothelial cell injury.