A retrospective cohort study was established of injecting drug users (IDUs) to assess evidence for hepatitis C virus (HCV) protective immunity through a comparison of incidence of initial HCV infection and HCV reinfection. Incidence of initial HCV infection was determined among HCV seronegative IDUs, and HCV reinfection determined among IDUs with newly acquired HCV infection, HCV viraemia and subsequent HCV RNA clearance. Serum was available for HCV RNA analysis from stored samples taken at the time of prior blood-borne virus screening. Potential HCV reinfection was defined as a positive HCV RNA following at least one negative HCV RNA. Incidence of initial HCV infection was 17/100 person-years (95% CI, 14-20/100 person-years). The incidence of potential HCV reinfection was 42/100 person-years (95% CI, 25-61/100 person-years), and after excluding cases without a change in HCV genotype and less than three consecutive HCV RNA negative assessment, incidence of reinfection was 31/100 person-years (95% CI, 17-62/100 person-years). Following adjustment for HCV risk behaviour variables the incidence rate ratio of HCV reinfection to initial infection was 1.11 (P = 0.8). Several cases of HCV reinfection appear to have developed persistent infection.