Cardiovascular problems associated with diabetes mellitus involves endothelial dysfunction in a mechanism that could be triggering by hyperglycemia (Ding et al., 2000). There is evidence that the endothelial dysfunction would be caused by an increase of reactive oxygen species (ROS) induced by high extracellular concentrations of D-glucose (Srinivasan et al., 2004). Protein kinases C (PKC) and p38MAPK could be involved as modulators of the synthesis of superoxide by NADPH oxidase (NOX), the main source of superoxide in human endothelium (Li et al., 2009). Our aim was to determine if the signaling pathway involves in regulation of NOX expression is dependent of PKC and/or p38MAPK activity. Biological samples were obtained from normal pregnancies (ethics committee approval and informed patient consent were obtained). Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion (37 Celsius degree) and cultured in medium 199 (M199) supplemented with 20% newborn and fetal calf sera. HUVEC were incubated (24 h) with M199 containing 5 mM (control) or 25 mM of D-glucose (high D-glucose) in absence or presence of calphostin C (CC, PKC inhibitor), PD169316 (p38MAPK inhibitor) and/or apocynin (NOX inhibitor). mRNA levels were determined by RT-PCR from total RNA using primers for NOX2, NOX4, p47phox, myeloperoxidase (MPO) and 28S. The ROS abundance was determined by fluorescence derivated from oxidation of dichlorofluorescein (DCF). High D-glucose increased (ANOVA unpaired Student’s t test, P<0.05, n=5-10) the mRNA levels of p47phox (0.5-fold), NOX2 (0.5-fold) and NOX4 (0.5-fold) in HUVEC. Increases of NOX2 and NOX4 mRNA levels were blocked by co-incubation with CC, PD169316 and apocynin. Co-incubation of CC and PD169316 do not show an additive response in presence of high D-glucose. Moreover, MPO was detected in HUVEC, which is a key protein in the inhibition mechanism of apocynin. As well as the case of mRNAs, high D-glucose increased the synthesis of ROS (2-fold), an effect blocked by co-incubation with CC, PD169316 and apocynin. In conclusion, high D-glucose increases the ROS synthesis in a mechanism mediated by an increase of the mRNA expression of NOX2 and NOX4. The mechanism underlying the transcriptional activation of NOX genes is related with a signaling pathway that involve PKC and p38MAPK. Also, high D-glucose increased the MPO expression, validating the use of apocynin like NOX inhibitor in HUVEC and, most importantly, showing a potential role of MPO in cellular responses to stress induced by high D-glucose.