Drug use in combination with psychiatric illness may lead to unsafe sexual risk behavior and increased risk for secondary HIV transmission among adolescents with HIV infection. We compared the prevalence of substance use for perinatally HIV-infected youth to uninfected adolescents living in families affected by HIV infection, and evaluated the association of psychiatric symptoms with risk of substance use. Among 299 adolescents (196 HIV+, 103 HIV-) aged 12-18 years enrolled in IMPAACT P1055, a multisite US cohort study, 14% reported substance use at enrollment (HIV+: 13%, HIV-: 16%). In adjusted logistic regression models, adolescents had significantly higher odds of substance use if they met symptom criteria for ADHD [adjusted odds ratio (aOR) = 2.7, Wald χ(2) = 5.18, P = 0.02], major depression or dysthymia (aOR = 4.0, Wald χ(2) = 7.36, P = 0.01), oppositional defiant disorder (aOR = 4.8, Wald χ(2) = 12.7, P = 0.001), or conduct disorder (aOR = 15.4, Wald χ(2) = 28.12, P = 0.001). Among HIV-infected youth, those with lower CD4 lymphocyte percentage (CD4% < 25%) had significantly increased risk of substance use (aOR = 2.7, Wald χ(2) = 4.79, P = 0.03). However, there was no overall association of substance use with HIV infection status, and the association between psychiatric symptoms and substance use did not differ by HIV status. Programs to prevent substance use should target both HIV-infected and uninfected adolescents living in families affected by HIV infection, particularly those with psychiatric symptoms.