Pyridoxamine, pyridoxine, and pyridoxal phosphate were tested to examine if they have antioxidant properties. Endothelial cells exposed to 0.5 mM H(2)O(2) for 2 hours increased the superoxide anion and lipid peroxide levels as biomarkers of oxidative stress. The increase of superoxide was mainly due to the activation of NADPH-oxidase by H(2)O(2). Preincubation of the endothelial cells with 0.1 or 1.0 mM of pyridoxamine or pyridoxal phosphate for one-half hour before H(2)O(2) exposure significantly reduced the superoxide and lipid peroxide compared to the cells exposed to H(2)O(2) only. Preincubation of the cells with 0.1 or 1.0 mM of pyridoxine also significantly reduced the lipid peroxide but did not significantly affect the superoxide level unless the preincubation time was extended to 24 hours. The prostacyclin release by endothelial cells was also significantly inhibited by H(2)O(2). However, the preincubation of endothelial cells with 1.0 mM of pyridoxamine, pyridoxine, or pyridoxal phosphate did not prevent that inhibition. These results indicate that pyridoxamine, pyridoxine, and pyridoxal phosphate acted as antioxidants and reduced the superoxide and lipid peroxides induced by H(2)O(2), but did not protect the cells from the effects directly related to H(2)O(2) itself.