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This study provides a test of General Strain Theory by examining the relations between strain, negative emotions, and biological hormones in the prediction of antisocial behavior across gender. Findings from a diverse sample of 512 undergraduate students reveal that strain and the ratio between testosterone to cortisol reactivity are associated with higher levels of antisocial behavior in males, but not females. In contrast, the effect of depressive symptoms on antisocial behavior is stronger at higher levels of strain and ratio of testosterone to cortisol reactivity in females. Drug use and depressive symptoms were found to partly mediate the association between strain and antisocial behavior in females, but not males.