Abstract Adolescence is a critical period for the development of a range of mental health conditions in females and males, particularly those marked by impairments in cognitive control. This research is focused on adolescent maturation of the mesocorticolimbic dopamine (DA) circuit, which is functionally linked to motivation, reward, and cognitive control. A unique feature of adolescent development of this circuit is the long-distance outgrowth of ventral tegmental area (VTA) DA axons from their prepubertal location in the nucleus accumbens (NAc) to the medial prefrontal cortex (mPFC), thereby strengthening connectivity between subcortical and cortical regions regulating cognitive control. The adolescent outgrowth of axons from the NAc to the mPFC coincides with the pubertal rise in gonadal steroid hormones, primarily estrogen in females and testosterone in males. Both hormones organize the nervous system during critical periods of development, including adolescence, resulting in structural and functional sex differences. The proposed studies will test the hypotheses that: Aim 1) gonadal hormones program the adolescent outgrowth of dopaminergic axons from the NAc to the mPFC; and Aim 2) adolescence is a sensitive period for hormone-dependent organization of the mesocorticolimbic circuit. A unique combination of virally mediated tract tracing techniques will be used to identify the synaptic varicosities of VTA neurons that extend their axons from the NAc to the mPFC during adolescence in female and male rats that either do or do not experience gonadal hormones during adolescence. The first hypothesis predicts that prepubertal gonadectomy will result in reduced DA innervation of the mPFC in both females and males and that hormone replacement (estrogen in females, testosterone in males) during adolescence will restore innervation. The second hypothesis predicts that hormone replacement in prepubertally gonadectomized rats in adulthood will not restore DA innervation of the mPFC. Because these exploratory studies predict hormonal effects in both females (estrogen) and males (testosterone), analyses will focus on within-sex comparisons of hormone status during adolescence or adulthood, rather than sex differences. This research will advance fundamental understanding of the developmental plasticity of the mesocorticolimbic dopamine circuit and how it is influenced by gonadal hormones in females and males. It will reveal not only how typical maturation occurs and within what developmental window(s) it can occur, but also how it can go awry (e.g., atypical timing of puberty onset). Knowing how typical maturation can go awry will provide novel insights into the developmental processes during puberty and adolescence that could lead to the emergence of psychopathology marked by deficits in cognitive control.