Abstract Serotonin (5-HT) action via the 5-HT2C receptor (5-HT2C R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT2C R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT2C R. The expression of 5-HT2C R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT2C R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT2C R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT2C R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT2C R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT2C R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.