Inward rectifying potassium (Kir) channels comprise a large family with diverse biophysical properties. A predominant feature of central nervous system (CNS) glia is their expression of Kir4.1, which as homomers are weakly rectifying channels, but form strongly rectifying channels as heteromers with Kir2.1. However, the extent of Kir2.1 expression and their association with Kir4.1 in glia throughout the CNS is unclear. We have examined this in astrocytes and oligodendrocytes of the mouse optic nerve, a typical CNS white matter tract. Western blot and immunocytochemistry demonstrates that optic nerve astrocytes and oligodendrocytes express Kir2.1 and that it co-localises with Kir4.1. Co-immunoprecipitation analysis provided further evidence that Kir2.1 associate with Kir4.1 and, moreover, Kir2.1 expression was significantly reduced in optic nerves and brains from Kir4.1 knock-out mice. In addition, optic nerve glia express Kir5.1, which may associate with Kir2.1 to form silent channels. Immunocytochemical and co-immunoprecipitation analyses indicate that Kir2.1 associate with Kir5.1 in optic nerve glia, but not in the brain. The results provide evidence that astrocytes and oligodendrocytes may express heteromeric Kir2.1/Kir4.1 and Kir2.1/Kir5.1 channels, together with homomeric Kir2.1 and Kir4.1 channels. In astrocytes, expression of multiple Kir channels is the biophysical substrate for the uptake and redistribution of K+ released during neuronal electrical activity known as ‘potassium spatial buffering’. Our findings suggest a similar potential role for the diverse Kir channels expressed by oligodendrocytes, which by way of their myelin sheaths are intimately associated with the sites of action potential propagation and axonal K+ release.