To analyze the process of mesenchymal differentiation in vitro, we examined 5 human glioblastomas as biopsy specimens, monolayer cultures and 3-dimensional fragment spheroid cultures for the immunohistochemical expression of extracellular matrix (ECM) components (collagen types I, III-VI, laminin) and integrin receptors (beta 1, beta 2, beta 3 and beta 4 chains). mRNA for type-I and type-IV collagen alpha I chains was quantified using reverse transcription-polymerase chain reaction. In situ, glioma cells expressed beta 1, the common beta chain of most integrin ECM receptors, while ECM components were restricted to vascular elements. Early monolayer cultures showed a marked increase in ECM components (interstitial collagens more than basement membrane components), and coexpression of ECM components and glial fibrillary acidic protein (GFAP) by most cells. beta 2 and beta 3 integrins were upregulated in the primary cultures. In the fifth passages, GFAP-positive cells were decreased and collagen-expressing cells increased. The spheroids exhibited preserved GFAP staining, neoexpression of beta 4 integrin in some tumors, and variable ECM expression by glioma cells which was lower than that in monolayer cultures. ECM deposition usually commenced in central spheroid areas where the Ki-67 proliferation index was low. We conclude that different culture systems are characterized by distinct expression patterns for ECM components and receptors, and that mesenchymal features in cultured gliomas arise due to transdifferentiation of glioma cells.