Methylglyoxal exerted a time and Gefitinib dose-dependent toxicity on cultured human brain endothelial cells; it significantly reduced the integrity of the barrier measured by both functional and morphological experiments. This is the first study to provide kinetic data on the toxicity of methylglyoxal by impedance-based cell electronic sensing, a noninvasive label-free technique. The two different cell viability assays we used were in complete agreement on the direct cellular damaging effect of methylglyoxal, impedance data reflecting changes in cell adhesion, cell shape and number were confirmed by MTT tests measuring the metabolic activity of cells. Our data lend support to and expand previous findings on the effect of methylglyoxal on human brain endothelial cells. We selected the human hCMEC/D3 cell line as a simplified model of the blood-brain barrier. This cell line is widely used in different experiments, including pharmacological and drug studies. To support the relevance of our data on hCMEC/D3 endothelial cell line, the effect of methylglyoxal was also tested on primary cultures of rat brain endothelial cells. The observed effects were in agreement with our observations on the human cell line, indicating a similar sensitivity of primary endothelial cells and hCMEC/D3 endothelial cell line for the toxic effects of methylglyoxal. We found no data on primary brain endothelial cells related to methylglyoxal in the literature, therefore the present observation is the first study to include primary brain endothelial cells in this setting. The relevance of our findings on endothelial cells is limited by the use of high concentrations of methylglyoxal to induce barrier damage, a common concern in cell culture studies. However, in four recent and independent studies both the time needed to measure methylglyoxal-induced injury in cultured endothelial cells and the concentrations used were in similar range as in our study. Damage by methylglyoxal is mediated not only via carbonyl stress, but also by oxidative stress. Reactive oxygen species are generated as by-products of protein glycation. Furthermore, methylglyoxal increases glycation of selected mitochondrial proteins resulting in increased formation of superoxide. Elevated level of ROS weakens the barrier integrity, however the contribution of methylglyoxal-triggered ROS production in the increased endothelial permeability is controversial. In the present study we confirmed that methylglyoxal treatment promotes oxidative stress in brain endothelial cells, similarly to previous studies on endothelial cells and other cellular systems. The kinetics of ROS production also helped to determine the optimal time point for protection assays and other experiments: a time point, where ROS formation was still elevated, was purposefully selected. In good agreement with the data from toxicity measurements methylglyoxal increased the permeability of human and rat brain endothelial monolayers. The effect was dose-dependent, with only high concentrations of methylglyoxal causing significant damage in barrier integrity.