Prior research suggests that the interconnections between substance use, HIV risk and lack of adherence to HIV medications are especially strong among homeless individuals. Thus, study of these interconnections warrants public health attention. The objectives of this paper are to describe patterns of alcohol and drug use, associations between substance use and participation in high-risk sex, and associations between substance use and adherence to HIV treatment regimens among a sample of 602 homeless or unstably housed HIV-seropositive individuals who are part of a housing-based intervention - the Housing and Health Study. Participants experienced high levels of substance use. Significant associations were found between substance use and adherence to HIV treatment medications, and between substance use and high-risk sexual practices within the entire group. Group analyses by sexual orientation/gender show that the association between substance use and treatment adherence is found primarily among heterosexual males whereas the relationship between several drugs and high-risk sexual practices is strongest among gay and bisexual men. Health professionals working with HIV-seropositive individuals should routinely ascertain housing status and screen for substance use and risky sex.