In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.