Na,K-ATPase is an integral membrane enzyme responsible for maintenance of the transmembrane Na+/K+ gradient which generates membrane excitability. Previous studies showed that oligodendrocytes within the CNS robustly expressed the alpha2 isoform of the Na,K-ATPase while oligodendrocytes in isolated cultures did not. We tested whether the levels of this isoform might be modulated by interactions with neurons. Western blots showed alpha2 protein expression was very low in rat optic nerve immediately after birth, but that expression was greatly increased by days 5 and 14. In adult optic nerves, levels were barely detectable. Since the first myelinated axons are observed in rat optic nerve at day 5, and the next 2 weeks are considered the period of peak myelination, this timing suggested a relationship between oligodendrocyte-neuron contact, myelination onset and the upregulation of the alpha2 isoform. In further experiments we plated oligodendrocytes in isolation or in co-culture with neurons dissociated from cerebral cortex at the day of birth. After 6 days in vitro, 45% of oligodendrocytes co-cultured with neurons expressed abundant alpha2 protein which was detected by immunohistochemistry, a six-fold increase over cells expressing alpha2 protein in isolated cultures. Conditioned medium from neuronal cultures did not affect alpha2 levels in oligodendrocytes. These results suggest that neurons may play a role in upregulating glial expression of the alpha2 isoform during peak periods of myelination, and that the effect is likely to be dependent on contact.