Tumor necrosis factor (TNF) exists in two bioactive forms, the membrane-integrated form and the proteolytically derived soluble cytokine. Cells that produce TNF are often responsive to TNF, allowing autocrine/juxtacrine feedback loops. However, whether the membrane form of TNF is involved in such regulatory circuits is unclear. Here we demonstrate that HeLa cells, expressing a permanently membrane-integrated mutant form of TNF, constitutively express TNF.TNF receptor complexes at their cell surface. These cells show a permanent activation of the transcription factor NF-kappaB, exert constitutive p38 mitogen-activated protein kinase activity, and produce high amounts of interleukin-6. In parallel, transmembrane TNF-expressing HeLa cells display high sensitivity to cycloheximide or interferon-gamma, similar to untransfected cells treated with these agents in combination with sTNF. Moreover, cycloheximide-induced apoptosis in transmembrane TNF transfectants can be blocked by the caspase inhibitor zVAD-fmk and does not necessarily need cell to cell contact, indicating a critical role of constitutive autotropic signaling of TNF.TNF receptor complexes. These data demonstrate that autotropic signaling loops of membrane TNF can exist, which may be of importance for cells that express both TNF and TNF receptors, such as T lymphocytes, macrophages, and endothelial cells.