To gauge the incidence of hepatitis C virus (HCV) infection and associated risk factors among inmates during their imprisonment, the authors recruited adult males in a long-stay Scottish prison into a cohort study between April 1999 and October 2000. On two occasions (at 0 and 6 months), saliva was collected for anonymous HCV antibody testing and risk behavior data were obtained through a self-administered questionnaire. The participation rate was 85% at both initial recruitment (612/719) and follow-up (375/441; 171 men were ineligible for follow-up). For inmates who reported never having injected drugs, ever having injected drugs, having injected drugs during follow-up, and having shared needles/syringes during follow-up, HCV incidences per 100 person-years of incarceration risk were 1, 12, 19, and 27, respectively. Ever having injected drugs (relative risk = 13.0, 95% confidence interval: 1.5, 114.3) and having shared needles/syringes during follow-up (relative risk = 9.0, 95% confidence interval: 1.1, 71.7) were significantly associated with HCV seroconversion. The effectiveness of existing interventions, including the provision of bleach tablets for sterilizing injection equipment, was suboptimal. The development of methadone maintenance programs in prisons and the creation of drug courts to keep offending drug injectors out of prison might help to reduce transmission in this setting.