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The present longitudinal study examined multiple biobehavioral measures, including heart rate, blood pressure, emotion, and salivary cortisol and salivary alpha amylase (sAA), in response to a stressor in 193 low-income 14- to 17-year-olds, half of whom were prenatally cocaine exposed (PCE). Youth's lifetime substance use was assessed with self-report, interview, and urine toxicology\/breathalyzer at Time 1 and at Time 2 (6\u201312 months later). PCE \u00d7 Gender interactions were found predicting anxiety, anger, and sadness responses to the stressor, with PCE girls showing heightened responses as compared to PCE boys on these indicators. Stress Response \u00d7 Gender interactions were found predicting Time 2 substance use in youth (controlling for Time 1 use) for sAA and sadness; for girls, heightened sadness responses predicted substance use, but for boys, dampened sAA responses predicted substance use. 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