This article presents the findings of a survey completed by 1351 predominantly Mexican American middle school students residing in a large urban center in the U.S. Southwest. The study explores possible associations between drug use attitudes and behaviors and gender (biological sex), gender identity, ethnicity, and acculturation status. Based on the concepts of "machismo" and "marianismo" that have been used to describe Mexican populations, four dimensions of gender identity were measured: aggressive masculinity, assertive masculinity, affective femininity, and submissive femininity. In explaining a variety of indicators of drug use behaviors and anti-drug norms, gender alone had limited explanatory power, while gender identity-often regardless of gender-was a better predictor. Aggressive masculinity was generally associated with higher risk of drug use, while the other three gender identity measures had selected protective effects. However, the impact of gender identity was strongly mediated by acculturation. Less acculturated Mexican American students reported lower aggressive masculinity scores than non-Latinos. Less acculturated Mexican American girls reported both the lowest aggressive masculinity scores and the highest submissive femininity scores. More acculturated Mexican American students, along with the less acculturated Mexican American boys, did not appear to be following a polarized approach to gender identity (machismo and marianismo) as was expected. The findings suggest that some aspects of culturally prescribed gender roles can have a protective effect against drug use behaviors and attitudes, possibly for both girls and boys.