β₂-Agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D₂ from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-⍺ (TNF-⍺), there is no information about their regulation by β₂-agonists. Thus given the importance of TNF-⍺ in inflammation and the widespread use of β₂-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) β₂-agonists on the secretion of TNF-⍺ from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-⍺ (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-⍺–sensitive cell line, WEHI-164, with an IC₅₀ of 71, 50, and 29 nmol/L, respectively. Specificity for β-adrenergic receptors was shown with propranolol. The inhibitory effect of β₂-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-⍺ release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, β₂-agonists did not show tachyphylaxis for the inhibition of TNF-⍺ release. Thus selective β₂-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-⍺ from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of β-agonists may be important in their mode of action in the treatment of allergic diseases. (J Allergy Clin Immunol 1997;100:825-31.)