Abstract Historically, women have been underrepresented in addiction research. Given the 7.2 million women affected by substance use disorder (SUD) in 2018, this lack of research contributes to a lack of effective treatment options. Exercise has recently been highlighted as a promising non-pharmacological treatment for SUD and women may be uniquely affected, but mechanisms for this phenomenon are not fully understood. Previous investigations in humans and rodent models have shown that both physical activity and preference for psychostimulant drugs (e.g., cocaine) correlate with fluctuations in estradiol (E2) throughout the menstrual cycle. Specifically, women report greater euphoria, desire, and energy after drug use during the follicular (i.e., higher E2) phase of their cycle, while blockade of estrogen receptors in female rats reduces self-administration of cocaine. In addition, E2-sufficient female rodents are more physically active, while loss of E2 in postmenopausal women and ovariectomized (OVX) rodents reduces physical activity. One potential mechanistic explanation for this is the effect of E2 on dopamine (DA) signaling in the nucleus accumbens (NAc), a brain region associated with reward and addiction. My previous work has shown a strong correlation between reduced physical activity in E2-depleted female rodents and 6-pyruvoyltetrahydropterin synthase (Pts), a gene upstream of DA synthesis in the NAc. This proposal seeks to compare the effect of E2 loss on voluntary wheel-running (VWR) and conditioned place preference (CPP) for cocaine in young (12-16 weeks) wild-type female C57Bl6/J mice that have undergone OVX operations. To determine mechanism, we propose to stereotaxically inject E2 directly into the NAc brain region of each group of OVX mice to determine the rescue effect on both VWR and CPP for cocaine. We will examine the potential mechanism by measuring changes in DA synthesis and receptor expression in the NAc following OVX and E2 treatment and associating such changes with each behavior. Aim 1 will test the hypothesis that OVX will reduce VWR in young female mice, while direct microinjection of E2 into the NAc will rescue this effect. We will test whether this behavioral change occurs by reduced DA synthesis and receptor expression in the NAc via a mechanism involving reduction in Pts. Aim 2 will test the hypothesis that OVX will reduce conditioned preference for cocaine, while direct microinjection of E2 into the NAc will increase conditioned preference. We will test whether this behavioral change occurs by a similar mechanism to VWR as tested in Aim 1. This proposal examines the effect of E2 on rewarding behaviors in female rodents and will test whether the mechanism involves DA synthesis in the NAc brain region. Furthermore, this project will provide the applicant valuable training, serving as a foundation for a future career in exercise and addiction research.