Interleukin 10 (IL-10) expression has been found to be correlated with the extent of malignancy in gliomas. In vitro, IL-10 increases proliferation and migratory capacity in human glioma cell lines. In this study, we localized the site of IL-10 synthesis in gliomas to cells of microglial origin. Biopsy specimens from 11 patients with malignant glioma were processed on native tissues and at early cell culture passages (0-4). IL-10 mRNA was analyzed by RT-PCR and in situ hybridization. Protein was quantitatively assessed by ELISA in cell culture supernatants, and cells expressing IL-10 were determined by a combination of immunohistochemistry for CD68 (specific for microglia/macrophage lineage) and IL-10 in situ hybridization. IL-10 mRNA decreased from passage 0 to 4 in all samples and was undetectable beyond passage 5. Such downregulation of mRNA leads to a steep decrease of IL-10 protein in culture supernatants (below detection level, 0.05 ng/ml, beyond passage 1). The combination of in situ hybridization for IL-10 and CD68 immunostaining revealed that only cells of the microglia/macrophage lineage produced IL-10 mRNA. Our results identify microglia/macrophage cells as the major source of IL-10 expression in gliomas which decreases markedly during early passages of primary cultures of human gliomas due to a progressive reduction of microglia/ macrophages present. Int.