Disruptions of connectivity in the medial prefrontal cortex (mPFC) have been linked clinical disorders including schizophrenia and ADHD. Evidence suggests that the mesocortical dopaminergic circuit, comprising projections from the ventral tegmental area (VTA) to the PFC, may be sensitive to disruption early in development. Progesterone receptor (PR) is transiently expressed in both the VTA and mPFC during critical developmental periods. PR within the VTA is expressed primarily in dopaminergic cells, as over 90% of PR immunoreactive cells are also immunopositive for tyrosine hydroxylase (TH). We have shown through retrograde tract tracing that many of the VTA projection cells that terminate in the mPFC express PR during development. Additionally, we have previously documented that inhibition of PR activity with RU486 during development reduced the density of TH-ir fibers in the prelimbic mPFC in rats. In the present study, utilizing both rats treated neonatally with RU486 (20mg/kg s.c.) and progesterone receptor knockout (PRKO) mice, we examined relative levels of TH protein in the VTA, and assessed cognitive flexibility performance using the attentional set-shift task and an adapted water-maze task. We found that in both RU486-treated rats and PRKO mice, there was a significant decrease in TH levels in the adult VTA compared to controls. Also, PR inhibition in both rats and mice led to a decrease in cognitive flexibility performance in adulthood. Collectively, these findings suggest that PR activity during development may play an important role in the maturationof the mesocortical dopaminergic pathway through actions in the VTA and/or in target cells of the mPFC, and may be critical for mPFC-mediated behaviors.