This longitudinal study examined how separation and family conflict mediated the effects of two acculturation variables (English language use and generational status) on substance use (alcohol, tobacco, and marijuana use) and deviant behavior outcomes in a Mexican American high school age sample. Structural equation modeling indicated that separation was a significant mediator of the relationship between acculturation and alcohol use, tobacco use, and deviant behavior. Family conflict mediated the effects of acculturation on marijuana use and deviant behavior. Model comparisons across gender groups suggested that generational status was an influential acculturation measure for females but not males. Additionally, English language use maintained a direct effect on marijuana use among females, whereas this relationship was mediated by separation for males.