Non-targeted effects induced by ionizing radiation have been demonstrated both in vitro and in vivo. Previously, we have also demonstrated the existence of non-targeted effects in intact Arabidopsis seeds following low-energy heavy-ion radiation. In the present study, 6.5 MeV protons with 8 × 10(11) ions/cm(2) and 2 × 10(11) ions/cm(2) fluence respectively were used to irradiate non-shielded or partial-shielded Arabidopsis seeds to further explore the mechanisms which regulate in vivo non-targeted effects and to investigate the difference between damage caused by non-targeted effects and direct irradiation. Results showed that excess reactive oxygen species (ROS) are present in the non-irradiated part of the partially irradiated samples, indicating that in vivo non-targeted effects can promote the generation of excess metabolic ROS in the non-irradiated shoot apical meristem/root apical meristem cells. Furthermore, pretreatment with 0.5% ROS scavenger dimethyl sulfoxide (DMSO) or 0.02 mM reactive nitrogen species (RNS) scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) significantly suppresses the non-targeted effects in the partially irradiated samples, while in the whole-body irradiated samples, the cPTIO pretreatment has no effect. On the other hand using antioxidant enzyme assays, superoxide dismutase activity was found to increase for partial irradiated samples and decrease for the whole-body exposed seeds. Taken together, these results implicate that damage caused by non-targeted effects is different from that induced by direct irradiation in vivo. Metabolic products such as ROS and RNS are involved in the in vivo non-targeted effects.