We have previously demonstrated that there is a factor present in some human serum which inhibits the passive cutaneous anaphylaxis reaction mediated by IgE. The present study analyzes the effect of this factor on mast cell IgE-dependent tumor necrosis factor (TNF)-alpha release. Rat peritoneal mast cells and RBL-2H3 cells treated with monoclonal mouse IgE anti-dinitrophenol (anti-DNP) followed by DNP-bovine serum albumin (DNP-BSA) were used and TNF-alpha release was measured at different time points. Similarly the percentage of rat peritoneal mast cell degranulation was determined. Results show a period of 30 min as optimal incubation time for TNF-alpha release in both mast cell populations. Human serum anaphylaxis inhibitory factor enriched fraction inhibited TNF-alpha release when it was in contact with IgE before the antigen treatment. Under these conditions the percentage of mast cell degranulation decreased. Mast cells incubated before IgE treatment with the factor alone do not release TNF-alpha and the percentage of degranulation increases due to a non-IgE-dependent process. A possible role of the inhibitory factor in the later phase reaction in addition to immediate hypersensitivity described previously is suggested.