The GABAergic system was investigated in C-6 astrocytoma cells and C-1300 neuroblastoma cells in culture and compared to that in mouse brain. The activities of glutamate decarboxylase, GABA-transaminase, succinic semialdehyde dehydrogenase and glutamate dehydrogenase were measured. In the cultured cells, only glutamate dehydrogenase activity was equal or greater than that of mouse cerebral cortex. Glutamate decarboxylase in both cell lines was 2%, while GABA-transaminase and succinic semialdehyde dehydrogenase activities were less than 20% of those found in brain. In spite of the disparate enzyme activities, GABA, glutamate, and α-ketoglutarate concentrations were similar in the cell lines and cerebral cortex. The anticonvulsant drugs sodium valproate and aminooxyacetic acid increased cortical GABA concentrations but either had no effect or decreased GABA in the cells in a complete medium. The convulsant isoniazid decreased GABA in mouse brain but had no effect in either cell line. In the absence of pyridoxal in the medium, some drug effects could be induced in the cultured cells. It is concluded that the differing responses of the GABAergic system in the mouse brain and cell lines may be attributed in part to the fact that the cells do not represent an integrated system and are of tumor origin.