Xerostomia frequently arises in patients with head and neck malignancies that are treated by radiation. However, the mechanisms responsible for the destruction of the salivary gland remain unknown. We previously established a xerostomia model of mice and identified the pathway through which nitric oxide (NO) affects the pathogenesis of radiation-induced salivary gland dysfunction. Although the toxicity of NO alone is modest, NO with superoxide anion (O 2•− ) rapidly forms peroxynitrite (ONOO − ), a more powerful toxic oxidant. In this study, we used the experimental model to examine: 1) when NO and O 2•− production is maximum in the salivary gland after irradiation; 2) whether peroxynitrite, as assessed by nitrotyrosine production, is responsible for salivary gland dysfunction; and 3) the effect of the iNOS selective inhibitor, aminoguanidine (AG), on nitrotyrosine formation. The increases in production of NO and O 2•− in the salivary gland peaked on day 7 after irradiation. Nitrotyrosine detected immunohistochemically was significantly reduced by AG in the salivary gland. On the basis of these results, we concluded that NO together with O 2•− forms the more reactive ONOO − , which might be an important pathogenic factor in radiation-induced salivary gland dysfunction.Radiotherapy is an established therapy for head and neck malignancies, but the treatment field might include normal several tissues and organs. As a result, xerostomia due to salivary gland dysfunction is a clinically important side effect that is often irreversible (23, 25). The actions of radiation include a direct effect of the radiation itself and the indirect DNA injury caused by the •OH radical, which induces cell death and salivary gland atrophy (3, 17). However, the actual pathogenesis of radiation-induced xerostomia is obscure, and an effective treatment has not been established.We previously established a xerostomia model using mice and identified the inflammatory pathway through which NO affects the pathogenesis of radiation-induced salivary gland dysfunction (27). NO is a radical molecule and has been identified as a vascular endothelium-derived relaxing factor (7,9,20). NO is generated by three types of nitric oxide synthase (NOS): neural NOS, endothelial NOS and inducible NOS (20). The expression of inducible NOS (iNOS) is induced by inflammatory cytokines (1,19) and that large amounts of NO are continuously released from activated macrophages through the NADPH-dependent oxidant deamination of L-arginine (9). The toxicity of NO is due both to NO itself and to NO-derived reactive oxidants (4). The modest toxicity of NO is increased by rapidly binding with NO together with O 2•− to form ONOO − , which is a more powerful, toxic oxidant (25). However, most of these reactive oxidants can not be directly detected in vivo. Thus, nitrotyrosine is used as a biomark-