PROJECT SUMMARY/ABSTRACT American Indian and Alaska Natives (AIANs) experience disproportionately high rates of lifetime substance use disorder with peak past-year prevalence rates at 16 years of age. AIAN adolescents ages 17 and younger have a higher prevalence and earlier initiation of drug and alcohol use compared with all other ethnic/racial groups in the US. Compared to other ethnic/racial groups, AIAN youth have the highest self-reported depression rates, and in 2014, suicide was the second leading cause of death for AIANs between the ages 10 and 34 years of age. These data demonstrate the need for early intervention to prevent substance use in AIAN adolescents. Cultural connectedness has been identified as a protective factor against substance use and depression for AIAN youth while promoting positive self-esteem and healthy identity formation. Culturally- grounded after-school programs (ASPs) use cultural values and practices as a basis for health promotion and disease prevention, a form of AIAN prevention. Culturally-grounded program differ from other cultural programs (culturally-based or culturally-adapted) because the development is guided or led by the local community, for the local community and are rooted in specific social and cultural contexts. This study builds on a 6-year partnership with ASPs serving AI adolescents to assess the impact of a 13-session culturally-grounded intervention, called Native Spirit (NS). NS is an ASP that is led by traditional knowledge holders with each session focusing on a community-specific cultural value and activity. NS aims to decrease and prevent substance use by enhancing protective factors, including strengthening cultural identity, self-esteem, and resilience. The goals of the proposed (R00) study are to: 1) strengthen self-esteem, resilience, and cultural identity, and 2) attenuate substance use among AI youth (ages 12-17) through participation in NS. This study will use a mixed methods waitlist control design to evaluate the impact of participation in the NS program. The study will measure changes to participant self-esteem, cultural identity, resilience, and substance use with a self-report survey at three timepoints and semi-structured interviews. This study provides an innovative connection between cultural engagement and substance use prevention for AI adolescents and also highlights unique opportunities for health promotion with collaborations with ASPs that serve AI communities. Through the MOSAIC K99/R00, I will gain training in qualitative and quantitative analysis, culturally-appropriate measurement of adolescent substance use, and the development and implementation of prevention interventions while transitioning to an independent investigator in a tenure-track faculty position. The resulting R00 phase research aligns with NIDA Strategic Goal 2 to “develop new and improved strategies to prevent drug use and its consequences” with AI communities in the Southwest.