To assess the relationships between HIV transmission risk behaviours, HIV serostatus and knowledge of HIV serostatus among active injection drug users (IDUs) residing in Tallinn, Estonia, we conducted HIV testing and administered a standardized interview to 266 participants reporting recent injection drug use. In total, 45% were HIV positive, and of those, 39% knew their HIV serostatus. Those who knew their HIV-positive serostatus were less likely to report giving someone else their needle after they used it (9%) than were HIV-negative participants (23%) and those who were HIV positive but unaware of their HIV-positive serostatus (25%). There were no statistically significant differences in unprotected sex or other drug use behaviours between the groups. Most participants reported that HIV can be transmitted through sharing needles (98%) and unprotected sexual activity (93%). Prevention needs of IDUs in this area include increasing the rates of HIV testing and implementing effective programmes to reduce sexual and drug use risk behaviours.