Chronic infection by Opisthorchis viverrini (OV) is a strong risk factor for developing cholangiocarcinoma (CCA). To clarify the involvement of oxidative stress and lipid peroxidation (LPO)-derived DNA damage, the excretion of LPO-derived etheno DNA adducts was measured in urine samples collected from healthy volunteers and OV-infected Thai subjects. 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) and 3,N(4)-etheno-2'-deoxycytidine (epsilondC) levels were quantified by immunoprecipitation/high-performance liquid chromatography/fluorescence detection and (32)P-postlabeling TLC. Excreted etheno adduct levels were related to indicators of inflammatory conditions [malondialdehyde (MDA) and nitrate/nitrite levels in urine and plasma alkaline phosphatase (ALP) activity]. Mean epsilondA and epsilondC levels were 3 to 4 times higher in urine of OV-infected patients; MDA, nitrate/nitrite, and ALP were also increased up to 2-fold. MDA and ALP were positively related to epsilondA excretion. Two months after a single dose of the antiparasitic drug Praziquantel, epsilondA and epsilondC concentrations in urine of OV-infected subjects were decreased; MDA, nitrate/nitrite, and ALP were concomitantly lowered. We conclude that chronic OV infection through oxidative/nitrative stress leads to increased urinary excretion of the etheno-bridged deoxyribonucleosides, reflecting high LPO-derived DNA damage in vivo. These promutagenic DNA etheno adducts in bile duct epithelial cells may increase the risk of OV-infected patients to later develop CCA. Urinary epsilondA and epsilondC levels should be explored (a) as noninvasive risk markers for developing opisthorchiasis-related CCA and (b) as promising biomarkers to assess the efficacy of preventive and therapeutic interventions.