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Malignant features in three glioma cell lines were studied in four defined media of various complexity. The cell lines D37MG, D54MG, and GaMG were able to grow in monolayer culture in all media examined, and as multicellular tumor spheroids in the two most nutrient-rich media. In the defined media, none of the cell lines were able to migrate in a migration assay on poly-D-lysine-coated plastic surfaces. Flow cytometric analysis of the GaMG cell line demonstrated no medium-dependent selection of subclones of glioma cells in spheroids cultured for 30 d. Morphological diversity of spheroids varied according to the supplementation of the media. The capacity of glioma cells to invade cellular rat brain aggregates was intact in the media examined. However, glioma migration was severely inhibited by the lack of specific serum components. This study demonstrates that glioma growth and invasion was heterogeneously preserved in the defined media used. Depending on the assay to be used in the study of glioma cell behavior, the degree of medium supplementation has to be considered.