Serotonin signaling influences many neural processes and is highly implicated in the etiology and treatment of depression. Understanding how in vivo elements regulate serotonin signaling may provide insight into serotonergic function in the depressed brain. This work presents a modification to fast-scan cyclic voltammetric methods that enables rapid, spatially resolved, and chemically selective measurement of in vivo serotonin signaling in the substantia nigra pars reticulata. We used this modified technique to investigate the dynamics of serotonin neurotransmission. Initial investigations found that serotonin release can be evoked by electrical stimulations in vivo, but it is greatly attenuated in comparison to serotonin evoked in brain slices. We hypothesized that feedback from intact inhibitory circuitry restricted serotonin signaling in vivo. A comparison of in vivo dopamine and serotonin release revealed a critical role for the serotonin transporter (SERT) in limiting serotonin signaling. This process is likely involved in the therapeutic effects of selective serotonin reuptake inhibitors (SSRIs), which are common pharmacotherapies for depression. Further studies found that acute SSRI treatment uniquely enhances serotonin signaling by disrupting inhibitory feedback to the dorsal raphe nucleus. We hypothesized that this action plays an important role in the 3-6 week lapse between SSRI treatment onset and clinical efficacy. Indeed, in a study of chronic SSRI treatment, we found that long-term reductions in SERT function result in enduring enhancement of serotonin signaling. In a healthy brain, this signaling is the product of dynamic, multi-systemic modulation, and SERT activity likely drives both ambient and transient serotonin concentrations in a coordinated, equilibrated manner. Our finding that the introduction of mild stress can significantly impact the effects of SSRIs implicates a more complex network of elements in the modulation of serotonin signaling. Future work will address how stress interacts with serotonergic function.