Tumor-necrosis-factor-alpha (TNFa) is a key mediator of cell death. Release of TNFa triggered by ischemia in the rat heart originated from cardiac mast cells. To uncover the role played by TACE in causing release, we used the human mast cell line HMC-1 and TACE-/- deficient mouse embryonic fibroblasts (MEF). After transient transfection of HMC-1 with plasmid pME18S-TNFa containing the human cDNA, the spontaneous release increased from <2 to 921 pg/ml (n=10) at 24h. The intracellular TNFa rose from 235 to 20219 pg/mg protein. Mock transfection (plasmid containing GFP) showed unchanged concentrations. For MEF there was no significant difference between the TACE+/+ and the TACE-/- fibroblasts: Spontaneous release amounted to <2 pg mouse TNFa/ml (ratTNFa-ELISA); 24h after transfection human TNFa in supernatant was 722 and 843 pg/ml for TACE+/+ and TACE-/-, resp. (n=4). The intracellular TNFaincreased from 9.0 (mouse) to 411 (human) pg/mg protein for TACE +/+ and from 8.2 (mouse) to 397 (human)pg/mg for TACE -/- MEF. WESTERN blot of the transfected cell lysates of HMC-1 showed the same 17kD band as recombinant TNF. In solution it was present as the trimer. Thus, processing of transfected TNFais similar to that leading to naive release. Because TACE deficiency has no effect, other proteases must be capable of converting pro-TNFa to 17kD TNFa and allow its shedding.