The present knowledge about the interaction between the extracellular matrix (ECM) and gliomas is mostly based on studies of permanent cell lines. Since such cultures have undergone an extensive clonal selection in vitro, the experimental results obtained may be quite different from those obtained from studies on true biopsy specimens. The present work demonstrates how different ECM components affect tumor cell migration from human glioblastoma specimens grown as biopsy sample spheroids. Biopsy specimens from 12 glioblastomas and 1 gemistocytic astrocytoma were included in this study. Spheroids were directly initiated from the biopsy specimens, and after 3-4 weeks in culture, they were used in a migration assay. A custom-made filtered medium, where the high molecular weight (>100 kDa) proteins were removed, was supplemented with the following ECM components: laminin, fibronectin, collagen type IV and vitronectin. The cell migration was negligible when spheroids were propagated in the filtered medium. The ECM components as well as complete DMEM evoked strong stimulatory effects on different biopsy specimens. Opposed to that observed earlier for permanent glioma cell lines, highly variable responses were observed between the different biopsy samples on the various ECM components. In general, correlation analyses revealed that specimens that were strongly stimulated by laminin were also stimulated strongly by fibronectin, collagen type IV and vitronectin. This suggests that the capacity to migrate as a response to ECM was confined more to each biopsy specimen than to any specific ECM component. Since biopsy sample spheroids, as original tumors, consist of different cell types, an immunohistochemical characterization of the migrating cells was also performed. Anti-glial fibrillary acidic protein (GFAP) staining revealed both GFAP-positive and -negative migrating cells. Immunostaining for von Willebrand factor and CD11b indicated that the migrating cells were neither endothelial nor microglial cells. This study, therefore, indicates that migratory responses of glioma biopsy specimens to different ECM components is much more heterogeneous than that observed earlier for cell lines. Furthermore, the presented findings support the notion that gliomas may utilize different cell surface receptors for their migration, depending on the cell substrates available.