Ecodevelopmental theory is a theoretical framework used to explain the interplay among risk and protective processes associated with HIV risk behaviors among adolescents. Although ecodevelopmentally based interventions have been found to be efficacious in preventing HIV risk behaviors among Hispanic youth, this theory has not yet been directly empirically tested through a basic research study in this population. The purpose of this cross-sectional study was to empirically evaluate an ecodevelopmentally based model using structural equation modeling, with substance use and early sex initiation as the two outcomes of the ecodevelopmental chain of relationships. The sample consisted of 586 Hispanic youth (M age = 13.6; SD = 0.75) and their primary caregivers living in Miami, Florida. Adolescent, parent, and teacher reports were used. The results provided strong support for the theoretical model. More specifically, the parent-adolescent acculturation gap is indirectly related both to early sex initiation and to adolescent substance use through family functioning, academic functioning, perceived peer sexual behavior, and perceived peer substance use. Additionally, parent's U.S. orientation is associated with adolescent substance use and adolescent sex initiation through social support for parents, parental stressors, family functioning, academic functioning, and perceived peer sexual behavior and substance use. These findings suggest that HIV risk behaviors may best be understood as associated with multiple and interrelated ecological determinants.