To evaluate the role of oxidative stress and aflatoxin exposure on risk of hepatocellular carcinoma (HCC), a case-control study nested within a large community-based cohort was conducted in Taiwan. Baseline urine samples, collected from a total of 74 incident HCC cases and 290 matched controls, were used to determine by enzyme-linked immunosorbent assays the level of urinary 15-F(2t)-isoprostane (15-F(2t)-IsoP), a biomarker of lipid peroxidation. These samples had been previously analyzed for urinary aflatoxin B(1) (AFB(1)) metabolites and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Pearson partial correlation coefficient analysis showed that urinary AFB(1) metabolites and 8-oxodG were significantly associated with the level of urinary 15-F(2t)-IsoP. After adjustment for potential confounding factors in a conditional logistic regression model, urinary 15-F(2t)-IsoP was significantly associated with risk of HCC [above versus below the mean odds ratio (OR) = 2.53, 95% confidence interval (CI) = 1.30-4.93]. Moreover, when compared with subjects in the lowest tertile of 15-F(2t)-IsoP, there was a trend of increasing risk of HCC (P(trend) = 0.0008), with adjusted ORs (95% CIs) of 3.87 (1.32-11.38) and 6.27 (2.17-18.13) for the second and third tertile, respectively. In addition, the combination of urinary 15-F(2t)-IsoP above the mean and chronic hepatitis B virus (HBV) infection resulted in an OR of 19.01 (95% CI = 6.67-54.17) compared with those with low urinary 15-F(2t)-IsoP and without HBV infection. These results suggest that elevated levels of urinary 15-F(2t)-IsoP may be related to increasing level of aflatoxin exposure and are associated with an increased risk of HCC.