Microglia are the primary central nervous system immune effector cells. Microglial activation is linked to interactions with extracellular cytokines and the extracellular matrix (ECM). Astrocytomas are characterized by their diffuse nature, which is regulated by insoluble ECM components produced by the tumor cells that are largely absent from normal central nervous system tissue. The present study examined the influence of astrocytoma (C6 rat glioma) insoluble matrix components on interferon-γ (IFN-γ)-mediated inducible nitric-oxide synthase (iNOS) induction in microglial cells. We found that IFN-γ-stimulated iNOS induction and nitric oxide release was greater in microglia cultured on C6 glioma cell-derived matrices compared with microglia cultured on primary rat astrocyte-derived matrices. Culture of microglia on C6 glioma cell-derived matrices also led to activation of STAT1, augmentation of IFN-γ-induced STAT-3 activation, and an increase in IFN-γ-activated site (GAS)-luciferase reporter activity. In addition, culture of microglia on C6 glioma cell-derived matrices activated NF-κB DNA binding activity and transcriptional activity. The results suggest that insoluble matrix components derived from malignant glioma cells can regulate microglia activation. These factors may include ECM components, such as fibronectin, collagen, laminin, vitronectin, and other nondiffusible compounds, and laminin seems to a critical regulator of this process. Microglia activation and subsequent brain inflammation may influence tumor growth, treatment, and metastasis. Better understanding of the regulation of microglial activation by astrocytoma-derived insoluble matrix components may be important in the development of immune-based treatment strategies against malignant brain tumors.