This study estimated prevalence of HIV risk behaviors and its association with substance use and mental health problems among adolescents in treatment. A pooled dataset of 9,519 adolescents admitted to substance abuse treatment programs between 2002 and 2006 was analyzed. HIV risk behaviors, substance use, and mental health problems were assessed at treatment intake. Sixty percent of adolescents were engaged in at least one sexual or needle use risk behavior in the year prior to entering treatment. Sex with multiple partners, sex under the influence of alcohol or drugs, and unprotected sex were the most prevalent HIV risk behaviors. Several gender differences were found for specific types of sexual and needle use behaviors. Adolescents with substance dependence or other comorbid mental health problems were at increased odds for HIV risk. Findings suggest treatment programs may benefit adolescents better by screening them consistently for HIV risk behaviors and incorporating tailored interventions.