PURPOSE To evaluate the role of 3D MR spectroscopic imaging (MRSI) as a tool for characterizing heterogeneity within a lesion in glioma patients. MATERIALS AND METHODS Forty-nine patients with newly diagnosed glioma were studied with 3D water-suppressed proton (1-H) MRSI. Signal intensities from choline (Cho), creatine (Cr), N-acytel aspartate (NAA), and lactate/lipid (LL) were estimated from the spectra. Regions of interest (ROIs) corresponding to the metabolic abnormalities were defined and compared with the anatomic lesions. RESULTS This study showed that the tumor burden measured with either the volumes of the metabolic abnormalities or the metabolic levels in the most abnormal voxels was correlated with the degree of malignancy of the tumor. The volumes of elevated Cho and decreased NAA were useful for distinguishing low-grade from high-grade lesions. The volume of abnormal LL was correlated with the existence of necrosis and with the volume of contrast-enhancing lesions in high-grade lesions. The differences in the volume of abnormal LL were also statistically significant between patients in each grade. CONCLUSION These 3D-MRSI data provide important additional information to conventional MRI for evaluating and characterizing gliomas.