Three-dimensional spherical aggregates of cells, grown from a permanent human malignant glioma cell line (multicellular GaMG spheroids) and from a human glioma biopsy (fragment spheroids), were investigated by 1H NMR spectroscopy. In addition, 1H NMR spectra of biopsy specimens immediately after explantation and of cell monolayers from primary passage and passage 5 were acquired and compared to those of fragment spheroids. By allowing tumor cells to grow in a three-dimensional arrangement, many biological characteristics of the original tumor in vivo are preserved. A technical procedure was established, indicating that spheroids are particularly suited for NMR spectroscopic studies. Well-resolved proton NMR spectra were obtained from homogeneously reaggregated as well as from fragment spheroids which were immobilized in agar. We found that fragment spheroids closely resemble characteristic spectral patterns of the corresponding tumor tissue in vitro, thus making such tissue available for 1H NMR spectroscopic measurements under easy to standardize tissue-culture conditions. Furthermore, the effect of altering glucose supply on metabolism and growth was studied with multicellular GaMG spheroids. The spectroscopic differences found between cell suspensions and multicellular spheroids indicate that GaMG spheroids produce large amounts of lactate and that they can adapt their metabolism depending on glucose supply similar to tumors in vivo.