Previous studies have suggested that the production of cathepsin S (CatS), a cysteine protease, was specifically induced in radiation-induced rat mammary tumors. In this study, we further investigate the mechanism by which CatS is induced by radiation and its function. Radiation induced production of CatS at both the mRNA and protein level, and increased its protease activity. In addition, these radiation induced changes occurred in a dose and time-dependent fashion. Agents such as bleomycin, As(2)O(3) and H(2)O(2), which produce reactive oxygen species (ROS), also induced CatS expression; however, other agents that damage DNA such as taxol and cisplatin did not. Additionally, treatment of the cells with the ROS scavengers, N-acetylcysteine and catalase, inhibited the radiation induced CatS expression. Furthermore, radiation-induced ROS was also involved in IFN-gamma production, which was responsible for radiation-mediated CatS expression. Moreover, electrophoretic mobility shift assay (EMSA) data obtained using an IFN-stimulated response element (ISRE) oligonucleotide revealed that IFN regulatory factor-1 (IRF1) was the critical transcriptional mediator of IFN-gamma-dependent CatS production after radiation. Finally, CatS overespression was found to induce radioresistance; however, knockdown of CatS resulted in the suppression of radioresistance. Taken together, the results of this study indicate that radiation induced CatS expression via ROS-IFN-gamma pathways, and that this increased expression may be involved in radioresistance.