Multinucleated giant cells (MGC) have been long recognized as a histopathologic feature of tuberculosis, yet little is known about the underlying mechanism of tubercle bacillus-induced formation of these fused macrophages. The main purpose of this study was to characterize cellular mechanisms involved in MGC formation of swine microglia, the resident macrophages of the brain, in cultures containing nonopsonized Mycobacterium bovis. Within 2 h of incubation. MGC were readily detected in these cultures by light and transmission electron microscopy. MGC formation was blocked by anti-CD14 and anti-CD18 antibodies and by thalidomide, a potent inhibitor of tumor necrosis factor-α (TNF-α) production by microglia. Also, TNF-α alone induced MGC formation. These findings suggest that two microglial cell receptors, CD14 and a β2 integrin, and the cytokine TNF-α participate in M. bovis-induced swine microglial MGC formation.