The objective of this study was to determine the level of immunity from Hepatitis B infection in an Australian population of drug and alcohol users, and the validity of self-reported immune status. A cross-sectional survey was employed of drug and alcohol users presenting for treatment, who were clients of De Paul House, a community-based drug and alcohol withdrawal service in Victoria. Outcome measures were: hepatitis B serology and personal reports of hepatitis B immunity. A total of 118 people were enrolled; 22% were injecting drug users and 48% reported past injecting drug use, while 55% were alcohol users; 51% had no general practitioner; 73% of participants were unaware of whether they were protected from hepatitis B infection, while 19.5% believed they were protected. However, serology demonstrated that 52.2% of those who believed they were protected were not immune. Only 21% of participants were immune. This is the first study in an Australian setting since the 1970s to examine the effectiveness of vaccination targeted to this population, and demonstrates low rates of immunity. New strategies are needed to deliver vaccination to this group. These will need to take account of the often chaotic nature of their lives, the poor validity of self-reported immunity, and the lack of primary care links.