This study is one of the first studies in the Czech Republic evaluating the extent of the HIV epidemic among drug users. Interview data on demographics, drug use, sexual practices, and HIV knowledge and risk perception were obtained from study participants and a saliva sample was obtained for an HIV antibody test. Although the HIV infection rate is low (0.2%), the prevalence of high risk behaviors, such as sharing injecting equipment and unsafe sexual practices, indicate a potential for rapid HIV spread in this high risk population. Preventive measures need to be targeted toward drug users, especially those of younger age and women.