AbstractThe expressions of a membrane form of TNF (m‐TNF) by human alveolar macrophages (AM) and autologous blood monocytes from healthy donors were examined. Upon lipopolysaccharide (LPS) stimulation, AM produced 26‐kDa TNF‐α on their cell surface. We designed a bioassay for measuring m‐TNF in which macrophages were fixed with paraformaldehyde after stimulation for 18 h, then m‐TNF activity was assessed as cytotoxicity of fixed macrophages on L929 cells. This assay was specific to m‐TNF because:1) nosoluble factors were contributed to the cytotoxicity of fixed AM,2)anti‐TNF‐α monoclonal antibody completely neutralized m‐TNF activity, and3)m‐TNF activity was not altered after low‐ pH or high‐salt treatment. On LPS stimulation, AM produced significant amounts of m‐TNF earlier than TNF‐α secretion. AM also expressed significant amounts of m‐TNF when stimulated with other bacterial components and their derivatives. Interleukin (IL)‐4 suppressed both m‐TNF production and TNF‐α secretion. p‐Toluene‐ sulfonyl‐L‐arginine methyl ester (TAME) inhibited specifically TNF‐α secretion and not m‐TNF expression. Although blood monocytes produced small amounts of m‐ TNF, monocyte‐derived macrophages showed enhanced m‐TNF after cultivation with GM‐CSF for 10 days. These findings indicate that m‐TNF is expressed as a step in the TNF‐α producing system, and suggest that m‐TNF may play important roles in exhibition of macrophage function in situ.