Reactive oxygen species (ROS) are capable of affecting neuronal cell function and structure. Here, we investigated the direct effects of hydrogen peroxide (H(2)O(2)), one of the ROS, on axonal transport in cultured mouse dorsal root ganglion neurons using video-enhanced microscopy. Treatment of neurons with the H(2)O(2) donor tert-butyl hydroperoxide (TBHP; 10 nM-1 mM) inhibited anterograde and retrograde movement of organelles in a time- and concentration-dependent manner. Mitochondria and lysosomes were clearly swollen by TBHP at 100 μM and 1 mM in association with complete and irreversible cessation of axonal transport. In contrast, cytoskeletal structures were apparently unchanged even at the highest TBHP concentration (1 mM). Lipid peroxides, detected by swallow-tailed perylene derivative fluorescence, were produced by TBHP in plasma membranes and more highly in organelle membranes. The TBHP-induced inhibition of axonal transport, lipid peroxide production, and organelle swelling were blocked by pretreatment with α-tocopherol (vitamin E, 1 mM). These results suggest that H(2)O(2) inhibited axonal transport via lipid peroxidation along with degenerative changes in organelles.