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An estimated 170 million people worldwide carry the hepatitis C virus (HCV), and in more developed countries the prevalence and incidence of HCV is particularly high among injecting drug users (IDUs). Spontaneous clearance of HCV infection and reinfection is well recognized but the level of protection against further infection conferred by HCV infection and clearance remains uncertain. We conducted a prospective study of HCV infection in IDUs recruited in Melbourne, Australia, using a much shorter median testing interval than in previous studies. Incidences of naive infection and reinfection were calculated by the person-year method and Cox proportional hazards regression used to adjust for covariates. A significantly higher HCV incidence rate was measured in previously infected IDUs (46.8% per year) compared with HCV-naive IDUs (15.5% per year). The hazard ratio for previously infected IDUs compared to HCV-naive IDUs, after adjustment for time-dependent covariates, was 2.54 (95% confidence interval, 1.11-5.78, P > |z| < 0.05). Viral persistence after reinfection appeared similar to that following naive infection. Conclusion: Our data suggest that HCV infection in IDUs is more likely following prior infection and clearance than in HCV-naive individuals, implying no increased immunity against further infection. This result has important implications for the future development of an HCV vaccine.