Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent stimulator of macrophages and neutrophils and plays a role in inflammatory diseases. In this article, we report that mouse brain-derived microvascular smooth muscle cells (SM) and endothelial cells (En) in coculture with splenocytes support the colony proliferation of immature granulocyte-macrophage-like (GM) cells. Unstimulated SM and En cells release GM-CSF as shown by ELISA assay and SM expresses mRNA for GM-CSF by polymerase chain reaction (PCR). Stimulation of SM and En by a nonspecific activator (lipopolysaccharide) results in upregulation of GM-CSF production. GM colonies cannot be grown on cultured astrocytes or on extracellular matrix alone prepared from smooth muscle or endothelium. However, colonies form on the extracellular matrix and on astrocytes, either in the presence of SM- or En-conditioned medium or after the addition of recombinant GM-CSF. The GM cells are positive for nonspecific esterase, peroxidase, and MAC-1 markers but are negative for FC gamma receptors and for Thy 1.2, CD8, CD4, MHC class II, and Asialo GM1 markers. These observations emphasize the possibility for active participation of brain microvasculature SM and En in acute inflammatory reactions of the central nervous system.