The medial prefrontal cortex (mPFC) is an important brain region, that controls a variety of behavioral and functional outputs. As an important step in characterizing mPFC functionality, in this paper we focus on chemically defining serotonin transmission in this area. We apply cutting-edge analytical methods, fast-scan cyclic voltammetry (FSCV) and fast-scan controlled adsorption cyclic voltammetry (FSCAV), pioneered in our laboratory, for the first real-time in vivo analysis of serotonin in the mPFC. In prior in vivo work in the substantia nigra, pars reticulata, we found that our sub-second measurements of a single evoked serotonin release were subject to two clearance mechanisms. These mechanisms were readily modeled via Uptake 1, mediated by the serotonin transporters (SERTs), and Uptake 2, mediated by monoamine transporters (dopamine transporters (DATs), norepinephrine transporters (NETs), and organic cation transporters (OCTs)). Here in the mPFC, for the first time to our knowledge, we observe two release events in response to a single stimulation of the medial forebrain bundle (MFB). Of particular note is that each response is tied to a discrete reuptake profile comprising both Uptake 1 and 2. We hypothesize that two distinct populations of serotonin axons traverse the MFB and terminate in different domains with specific reuptake profiles. We test and confirm this hypothesis using a multifaceted pharmacological, histological and mathematical approach. We thus present evidence for a highly elaborate biochemical organization that regulates serotonin chemistry in the mPFC. This knowledge provides a solid foundation on which to base future studies of the involvement of the mPFC in brain function and behavior. To read more, click here.