The association between Opisthorchis viverrini infection and hepatobiliary disease was studied in northeastern Thailand. Positive rates of O. viverrini infection and antibody titers to O. viverrini adult worm extracts, which were determined by enzyme-linked immunosorbent assay (ELISA), were compared among four groups: Thai patients with cholangiocarcinomas (CHCA), those with calculus cholecystitis (CCLT), endemic area victims of traffic accidents, and Japanese individuals used as negative controls. While no difference was observed between cases with CCLT and the accident victims, the CHCA group showed a significantly higher positivity rate for fluke infection and higher antibody titers than the other groups. Comparison of the positivity rates and ELISA titers among intrahepatic and extrahepatic CHCA subgroups showed that only females with intrahepatic tumors had a low positive rate and significantly lower titers, with no other significant variation being evident. From these observations, a strong association between high intensity of past and/or present O. viverrini infection and the genesis of CHCAs was apparent in the majority of cases. However, in addition to the fluke infection, other unknown exogenous or endogenous factor(s) were suggested in the female cases.