NMR-visible lipid signals detected in vivo by 1H MRS are associated with tumour aggression and believed to arise from cytoplasmic lipid droplets. High-resolution magic angle spinning (HRMAS) 1H MRS and Nile Red staining were performed on human brain tumour biopsy specimens to investigate how NMR-visible lipid signals relate to viable cells and levels of necrosis across different grades of glioma. Presaturation spectra were acquired from 24 adult human astrocytoma biopsy samples of grades II (8), III (2) and IV (14) using HRMAS 1H MRS and quantified using LCModel to determine lipid concentrations. Each biopsy sample was then refrozen, cryostat sectioned, and stained with Nile Red, to determine the number of lipid droplets and droplet size distribution, and with Haematoxylin and Eosin, to determine cell density and percentage necrosis. A strong correlation (R=0.92, P<0.0001) was found between the number of Nile Red-stained droplets and the approximately 1.3 ppm lipid proton concentration by 1H MRS. Droplet sizes ranged from 1 to 10 microm in diameter, and the size distribution was constant independent of tumour grade. In the non-necrotic biopsy samples, the number of lipid droplets correlated with cell density, whereas in the necrotic samples, there were greater numbers of droplets that showed a positive correlation with percentage necrosis. The correlation between 1H MRS lipid signals and number of Nile Red-stained droplets, and the presence of lipid droplets in the non-necrotic biopsy specimens provide good evidence that the in vivo NMR-visible lipid signals are cytoplasmic in origin and that formation of lipid droplets precedes necrosis.