The content and distribution of the membrane-bound enzyme (Na+ + K+)-ATPase in a rat cerebral C6 glioma was determined by immunocytochemistry, immunoblots and enzyme assay. In the C6 glioma cell culture (Na+ + K+)-ATPase activity was about 20% of (Mg2+ + Na+ + K+)-ATPase activity. However, (Mg2+ + Na+ + K+)-ATPase activity in the cerebral C6 gliomas was very close to Mg2+ baseline and not significantly increased by Na+ and K+. As shown by immunoblotting, (Na+ + K+)-ATPase catalytic subunit was detected in excised samples of control cerebrum and as a trace in the intracerebral portions of C6 glioma but not at all in the extracranial portions of C6 glioma or in C6 glioma cell culture. (Na+ + K+)-ATPase was not detected immunocytochemically in paraffin sections of the extracranial or intracerebral portions of rat cerebral C6 glioma. The absence of staining for (Na+ + K+)-ATPase clearly demarcated projections of glioma within normal brain. These results suggest that C6 glioma has little if any expression of (Na+ + K+)-ATPase in vitro or in vivo. The small amount of enzyme epitope in the intracerebral portions represents contamination by normal cerebrum in the extracts.