To observe whether the impairment of endothelial nitric oxide synthase (eNOS) activity by elevated level of free fatty acid (FFA) is associated with activation of protein kinase C (PKC).The cultured human umbilical vein endothelial cells were used for four groups: control group, oleic acid (OA) group (10, 50, 100, 150, 200 micromol/L), GFX group and OA+GFX group. The activity of eNOS in each group was examined, and the activity and expression of PKC were determined in both control and OA groups. The method of immunocytochemistry was performed to detect the expression of p-eNOS(1177) in control, OA (100 micromol/L), GFX and OA+GFX groups respectively.Compared with control group, the eNOS activity of endothelial cells in OA group was significantly reduced in a dose-dependent manner, and the expression of p-eNOS(1177) were also impaired (0.0854 +/- 0.017 vs. 0.0134 +/- 0.023, P < 0.05). PKC transposition from endochylema to cytomembrane was observed by confocal microscopy. The PKC activity was degraded in endochylema, while enhanced in cell membrane inversely, and the gross activity of PKC increased after all. When GFX, an inhibition of PKC, was added to endothelial cells at the present of OA or not, the impaired eNOS activity were partly improved.Elevated level of FFA (here is oleic acid) leading to endothelial dysfunction results from a loss of eNOS activity and its serine phosphorylation, and it may be related to activation of PKC.