Conditioned medium (CM) from concanavalin A-activated mouse spleen cells inhibits antigen-induced 5-hydroxytryptamine (5-HT) release from mouse peritoneal mast cells when added for 24 or 48 h to cell cultures containing monoclonal IgE sensitizing antibody. We present the following evidence that the spleen cell-derived inhibitory factor is interferon-gamma (IFN-gamma): (a) CM from Con A-activated spleen cells and from Chinese hamster ovary (CHO) cells transfected with the murine (Mu)IFN-gamma gene both produced a graded inhibition of 5-HT release when added to peritoneal cell cultures containing IgE and subsequently challenged with antigen; (b) the relative inhibitory potency of the two preparations corresponded to their relative IFN content as determined by antiviral bioassay; (c) the inhibitory activity of spleen cell CM and of CM from IFN-gamma gene-transfected CHO cells was abolished by treatment with a rat monoclonal MuIFN-gamma-neutralizing antibody, i.e. 5-HT release returned to control levels, and (d) a highly immunopurified preparation of recombinant MuIFN-gamma retained the inhibitory activity, and was active at concentrations as low as 2 U/ml. The inhibitory activity of IFN-gamma appeared to be a direct effect of the cytokine on mast cells, since CM generated for different time periods from unfractionated peritoneal cells treated with purified recombinant MuIFN-gamma contained no inhibitory activity other than IFN-gamma itself, as demonstrated by neutralization of the cytokine with monoclonal antibody. Furthermore, purified recombinant MuIFN-gamma was equally active on mast cells enriched by density centrifugation up to 95% purity as on unfractionated peritoneal cells (1%-2% mast cells). Purified recombinant MuIFN-gamma inhibited 5-HT release induced by a range of different cell activators, namely antigen, anti-IgE, compound 48/80 and calcium ionophore A23187. Hence inhibition is not specific to IgE-dependent activation of mast cells, but seems to be directed more generally at the secretory process of these cells.