A number of neural and nonneural tumor cell lines of rat and human origin were assayed for neuron-specific enolase (NSE) by radioimmunoassay. Most neural tumor cell lines had appreciably higher levels of NSE than did the nonneural tumor cell lines, the highest levels being found in two anaplastic rat glioma lines ( F98 and T24). These two lines contained more than twice the amount of NSE found in a rat pheochromocytoma line (PC12) and in neuroblastoma lines derived from rats ( B35 and B50 ) or humans (IMR-32 and SHSY - 5Y ). Several of the rat glioma and schwannoma lines were inoculated intracerebrally into syngeneic rats. In the resulting tumors, NSE was demonstrable by immunohistochemistry only in those from the F98 and T24 cell lines. A number of ethylnitrosourea-induced rat tumors were also examined immunohistochemically for NSE: NSE was demonstrated in three anaplastic gliomas; three astrocytomas; and two mixed gliomas. Reactive astrocytes were also positive. Fibroadenomas of apocrine and mammary glands in rats were weakly positive, but other extraneural tumors tested were negative. Since normal neuronal elements, axonal swellings, and amine precursor uptake and decarboxylation cells are strongly positive for NSE, whereas glia and most other normal cells are negative, we hypothesize that the elevated metabolic demands imposed on neoplastic and reactive glial cells and on some extraneural tumors necessitate the opening up of metabolic pathways that are normally operative only in neurons and neuroendocrine cells, therefore resulting in the synthesis of the more stable neuron-specific form of enolase.