Voluntary wheel running is rewarding and believed to activate the same brain reward system as in alcohol and drug addiction. Brain-derived neurotrophic factor (BDNF), a well-known growth factor widely expressed in the brain, is modulated by both voluntary exercise and alcohol consumption. The aim of this study was to evaluate how voluntary exercise affects the expression levels of BDNF and its receptor TrkB in brain regions involved in positive and negative reinforcement. Additionally we wanted to evaluate the effect it may have on alcohol drinking behaviors in C57BL/6 mice, a mouse model which are naturally prone for engaging in voluntary exercise and voluntary alcohol consumption. We found a small upregulation in DG and CA1 after three weeks of exercise, confirming findings by others, and a significant 3-fold downregulation of BDNF in NAc after both three weeks of exercise and exercise followed by a five week period of either ethanol intake or not. Interestingly, we here show a significant 100-fold increase in BDNF after exercise and a 120-fold increase after both exercise and alcohol consumption in amygdala, a region involved in regulation of anxiety-related behavior and negative reinforcement. Additionally a slightly lower 10-fold increase in BDNF was seen after exercise and a 15-fold increase after exercise followed by ethanol in prefrontal cortex, a structure contributing to reward-related behavior. Behaviorally, we could not either directly following exercise or at five weeks post-exercise detect any significant effect of wheel-running on depression-related behavior. However, we did find that exercise significantly increased the alcohol intake.