Tumor necrosis factor-alpha (TNF alpha) protein and messenger (m)RNA distribution was studied in biopsy samples of glial brain tumors, using immunohistochemistry and in situ hybridization with molecular probes, to investigate the role of this cytokine in tumor proliferation and immunological host defense. Focal expression of TNF alpha was detected in four of four glioblastomas, one of two anaplastic astrocytomas, and four of five low-grade astrocytomas, regardless of their subtype or grade of malignancy, but in none of the normal peritumoral brain tissues used as controls. The TNF alpha protein and mRNA were present in reactive astrocytes and protoplasmic tumor cells, confined to areas of leukocyte or T-lymphocyte infiltrating, and less pronounced in tumor cells at the edge of necrosis. Additionally, TNF alpha reactivity was found in infiltrating macrophages and perivascular microglia. Immunohistochemistry and in situ hybridization for TNF alpha showed comparable reaction patterns and numbers of TNF alpha-positive cells, even though the sensitivity of in situ hybridization was significantly higher. Quantitative evaluation of TNF alpha protein, TNF alpha mRNA, and leukocyte infiltration revealed a significant positive correlation between the TNF alpha-positive reactive astrocytes and the number of lymphocytes present in corresponding areas. Together, these data lead to the conclusion that TNF alpha in reactive astrocytes and monocytic cells within tumor areas of high leukocyte infiltration and in tumor cells at the border of necrosis may represent one defense pathway of the immune system against tumor proliferation.