The effect of chemotherapy against glioma in mouse was evaluated by 31P NMR spectroscopy and flow cytometry. We found that administration of ACNU or tegafur at a dose less than LD50 resulted in the partial suppression of the ratio of inorganic phosphate (Pi)/phosphocreatine (PCr) and phosphomonoester (PME)/creatine phosphate (PCr) after 24 or 48 hr, although these ratios are usually increased together with growth of tumors. Flow cytometric analysis of glioma in vivo showed an accumulation in cells containing tetraploid DNA by G2M block 24-48 hr after treatment. However, the change occurred at a period slightly later than that of the Pi/PCr ratio. In contrast, histological change was noted at eight days after administration. Hence, it is concluded that in vivo 31P NMR spectroscopy can detect a change in metabolic pathways in tumors as early as 24-48 hr after the administration of chemotherapeutic agents.