Total lactic dehydrogenase (LDH) levels and LDH isozyme patterns were measured in homogenates of 12 autochthonous ethylnitrosourea-induced rat gliomas and 5 cloned rat astrocytoma cell lines maintained in culture and transplanted to brain or flank sites in syngeneic hosts. The total LDH values in the autochthonous gliomas did not differ appreciably from normal brain controls, but the proportions of the cathodal isozymes, LDH4 and LDH5, were increased to a degree similar to that reported by others in spontaneous human malignant astrocytic gliomas. The cloned astrocytoma lines, both in vitro and in transplants at intracerebral or subcutaneous sites, commonly demonstrated elevated total LDH values and, without exception, showed a preponderance of isozymes, LDH4 and LDH5, that was distinctly more marked than in autochthonous tumors. Especially in cultured and transplanted rat gliomas, these findings suggest that astrocytic tumor cells maintain energy supplies by utilizing anaerobic glycolysis in relatively hypoxic environments. These data further underscore the need to develop laboratory brain tumor models for use in therapy trials that not only retain the convenience and predictability of transplantable gliomas but also approximate closely the metabolic properties of human spontaneous gliomas.