The convulsant methionine sulfoximine is a potent glycogenic agent in the central nervous system of rodents in vivo. This investigation was undertaken to look for the basic mechanism underlying this property. Astrocytes were cultivated from newborn rat neopallium and glycogen was studied by both biochemical and ultrastructural methods. When the astrocytes were incubated in a medium containing 5.55 mM glucose, methionine sulfoximine (0.55 mM) induced a significant increase in their glycogen content. Glucose content did not change in astrocytes, but it diminished in the medium in all cases. When the decrease in glucose level in the medium was limited, the same glycogenic effects of methionine sulfoximine were observed, but the glycogen contents were higher. The augmentation of the concentration of the convulsant enhanced its glycogenic effect, but this was not directly dose dependent. When the flat and polygonal astrocytes were transformed into process-bearing astrocytes by dibutyryl cyclic AMP methionine sulfoximine always induced an increase in glycogen content. In this case, the values of glycogen contents were lower. In electron microscopy, no glycogen particles were present in the astrocytes even after methionine sulfoximine treatment, contrary to the case in vivo. These results show that the convulsant does not need the presence of neuronal cells to induce glycogen accumulation and that astrocytes may be the direct cell targets. The apparent discrepancy between the biochemical and ultrastructural data is probably due to the relatively low concentration of glycogen in cultured astrocytes.