PROJECT ABSTRACT Headache has been one of the top three causes of disability adjusted life years for the past three decades among adolescents and young adults. The inspiration for this research comes from the disproportionately higher burden that falls on girls and women starting in puberty, when prevalence rises more quickly in girls than boys, then continues to be higher in women throughout the middle years of life. Puberty also appears to be a sensitive period in brain development when sex differences in regions key to headache such as the amygdala are driven by pubertal hormones. Despite these sex differences, the role of pubertal hormones such as estrogen and testosterone are not yet well-enough understood to be targets for prevention. This proposal is focused on the innovative study of gender affirming hormone therapy with testosterone in transmasculine adolescents as a model of puberty in which the timing of estrogen and testosterone changes are controlled. The aims of this proposal are to 1) determine association between gender affirming hormone therapy with testosterone in adolescence and headache in a cross-sectional study from in a multi-center health record database, 2) determine change in headache burden in response to gender affirming hormone therapy with testosterone and 3) determine changes in brain structure and function in response to gender affirming hormone therapy with testosterone. The scientific objective of this proposal is to start to outline the key points in development at which pubertal hormones can alter risk for headache and the physiologic underpinning for this alteration. These targets can then be applied in future clinical studies of hormonal management of headache in adolescence. This is a five-year career development research proposal. I am currently an Assistant Professor at the University of Colorado School of Medicine’s Department of Pediatrics in the Division of Child Neurology at Children’s Hospital Colorado. The outlined proposal builds on my previous research and clinical experience in neuroimaging of pubertal brain development and pediatric headache by expanding to a model of transgender adolescent headache. The proposed research and training plan will prepare me with a unique skillset for translational research as an independent clinician scientist in the emerging field of the effect of sex steroids on neurologic conditions in transgender youth and in headache sex differences research.