AIMS: This study was aimed at determining the seroprevalence of hepatitis C virus (HCV) infection in Romania and the possible risk factors and modality of HCV transmission. METHODS: A nationwide cross-sectional survey among the adult population was conducted between 2006-2008 in Romania through a population multicenter stratified random cluster sampling. Serum samples from 13,460 subjects were tested with a 3rd generation ELISA and a standardized questionnaire concerning the socio-demographic characteristics and potential risk factors was used. RESULTS: The prevalence rate of HCV infection in Romanian adult population was 3.23% with significant differences between the main geographical regions (Moldavia 4.25%, Wallachia & Dobrogea 3.35% and Transylvania & Banat 2.63%), as well as between different counties (maximum 7.19%, minimum 0.56%). Overall participation rate to the survey of the selected subjects was 74.69%. Risk factors for HCV infection were: blood/blood products transfusions (p=0.0001), previous surgery (elective and emergency, p=0.0001 and p=0.043, respectively), frequent hospitalizations (p=0.0001), injections at home (p=0.0001), accidents/trauma (p=0.0001), occupational hazard related to blood exposure (p=0.025), intravenous drug administration (p=0.002), a partner chronically infected with HCV/hepatitis B virus (HBV) (p=0.046), first sexual intercourse <18 years (p=0.019), familial exposure to HCV/HBV infection (p=0.001) or to chronic HBV/HCV liver disease (p=0.001), personal history of chronic HBV infection (p=0.001). HCV RNA positivity was detected in 91% of the anti HCV positive subjects. CONCLUSIONS: Overall HCV prevalence in Romania is 3.23%. Both nosocomial and non-nosocomial routes are implicated as risk factors for HCV infection.