The objective of this study was to assess differences in HIV, hepatitis B and hepatitis C seroprevalence among injecting drug users (IDU) in four Australian cities. 872 current IDU were recruited in approximately equal numbers from each of Adelaide, Melbourne, Perth and Sydney, and interviewed individually using a structured questionnaire. Fingerprick blood samples were taken from the majority of respondents, and tested for past exposure to the three viruses. HIV and hepatitis B and C raw seroprevalences were compared across cities, and comparisons were made of age-standardised seroprevalences for hepatitis B and C. Three percent of all respondents were HIV seropositive; 19% (23% age-standardised) were hepatitis B seropositive and 55% (60% age-standardised) were hepatitis C seropositive. There were general city differences and gender, sexual preference and treatment status group differences between the cities. Sydney respondents had the highest risk of infection for all three viruses in all comparisons. This was particularly striking for HIV among non-heterosexual men. Various explanations for the findings were considered, including city differences in demographic and drug use variables, underlying patterns of risk behaviour, and period/cohort effects. It was concluded that none of these explanations appeared to fit the pattern of findings, and that these probably represented true underlying differences in size of pools of infection. The reasons for this, however, cannot be ascertained from this study.