Tumor necrosis factor-alpha (TNF-a)-inducing activity of several mycoplasmas including Mycoplasma pneumoniae, a causative agent in human respiratory infectious diseases, was investigated. Purified peritoneal macrophages from BALB/c mice markedly enhanced their cytotoxic activity to Meth A cells, when cultured with either viable or non-viable mycoplasmas. The supernatants of the macrophage culture with mycoplasmas, M. pneumoniae and Acholeplasma laidlawii, showed the potent cytotoxic activity to TNF-a-sensitive L cells but not to TNF-a-insensitive L cells. Addition of anti-TNF-a antiserum inhibited completely the cytotoxic activity of these supernatants, indicating that a major part of the cytotoxic activity might be due to TNF-a. Various other mycoplasmas, either glucose-or arginineutilizing species, as far as tested showed also the potent activity to produce TNF-a. These results strongly suggest the possibility that mycoplasmas possess the activity of TNF-a induction which might be responsible for a part of enhancement of cytotoxic activity of macrophages and resistance to infection with mycoplasmas in vivo.Mycoplasmas have been isolated from almost all domestic and laboratory animals and man, and often cause the mycoplasma diseases such as respiratory infections and others. In humans, Mycoplasma pneumoniae is the causative agent of upper respiratory tract infections and pneumonia (14). These microorganisms lack the specific cell wall components of bacteria, such as diaminopimelic and muramic acids, and exist predominantly in an extracellular environment in close association with the cell plasma membrane (5). Although mycoplasmas can cause minimal morphological changes in mycoplasma-infected cell cultures, the possible simultaneous effects are numerous, in mycoplasma-infected immunocytes, such as lymphocytes, macrophages, and hybridoma cell lines, including enhancement of productions of immunoglobulin (26), cytokines (23), and interferon (4, 10, 24). Furthermore, the cytotoxicity of macrophages (17,20) and natural killer (NK) cells (7,18) to target cells are enhanced, when these cell lines were contaminated with mycoplasmas. Although the mechanisms of enhancement of macrophages-mediated 231