Our aim was to compare the prevalence of antibody to hepatitis C virus (anti-HCV) among recently initiated injecting drug users (IDUs) in London and Glasgow, and to identify risk factors which could explain differences in prevalence between the cities. Complementary studies of community recruited IDUs who had initiated injection drug use since 1996 were conducted during 2001-2002. Data on HCV risk behaviours were gathered using structured questionnaires with identical core questions and respondents were asked to provide an oral fluid specimen which was tested anonymously for anti-HCV but was linked to the questionnaire. Sensitivities of the anti-HCV assays for oral fluid were 92-96%. Prevalence of anti-HCV was 35% (122/354) in London and 57% (207/366) in Glasgow (P < 0.001). Multifactorially, factors significantly associated with raised odds of anti-HCV positivity were increasing length of injecting career, daily injection, polydrug use, having had a needlestick injury, and having served a prison sentence. In addition lower odds of anti-HCV positivity were associated with non-injection use of crack cocaine and recruitment from drug agencies. After adjustment for these factors, the increased odds of anti-HCV associated with being a Glasgow IDU were diminished but remained significant. HCV continues to be transmitted among the IDU population of both cities at high rates despite the availability of syringe exchange and methadone maintenance. Effectiveness of harm reduction interventions may be compromised by inadequate coverage and failure to reduce sufficiently the frequency of sharing different types of injecting equipment, as well as the high background prevalence of HCV, and its high infectivity. Comprehensive action is urgently required to reduce the incidence of HCV among injectors.