G-protein regulated inward-rectifier potassium channel 2 (GIRK2) is reported to be expressed only within certain dopamine neurons of the substantia nigra (SN), although very limited data are available in humans. We examined the localization of GIRK2 in the SN and adjacent ventral tegmental area (VTA) of humans and mice using either neuromelanin pigment or immunolabeling with tyrosine hydroxylase (TH) or calbindin. Serial transverse 50µm sections were cut from formalin-fixed brainstems of five brain donors free from significant abnormalities (aged 85±3 years old) and compared with 40µm sections from paraformaldehyde-fixed brains from six adult C57BL6/J mice. GIRK2 immunoreactivity was found in nearly every human pigmented neuron or mouse TH-immunoreactive neuron in both the SN and VTA, although considerable variability in the intensity of GIRK2 staining was observed. The relative intensity of GIRK2 immunoreactivity was determined using Image J software grayscaling and the proportion of TH-immunoreactive neurons containing strong GIRK2 immunoreactivity determined (>50% of comparator intensity). In both species, nearly all SN TH-immunoreactive neurons had strong GIRK2 immunoreactivity compared with only 50-60% of VTA neurons. Most paranigral neurons also contained calbindin immunoreactivity and approximately 25% of these and nearby VTA neurons also had strong GIRK2 immunoreactivity. These data show that high amounts of GIRK2 protein are found in most SN neurons as well as in a proportion of nearby VTA neurons, and that previous studies suggesting GIRK2 is located only in limited neuronal groups within the SN of humans and mice have erroneously included VTA regions as part of the SN.