Upon activation by most stimuli, basophils and human lung mast cells simultaneously release histamine and arachidonic acid metabolites. Hyperosmolar activation was examined, and both cell types were shown to release histamine but little or no leukotriene C4 or D4 (LTC4/D4) and, in the case of mast cells, little or no prostaglandin D2 (PGD2). In addition, hyperosmolar buffers were capable of preventing the formation of LTC4/LTD4 in basophils stimulated by anti-IgE when added simultaneously, or 2, 5, or 10 min after, the addition of anti-IgE. Catabolism of PGD2 and LTC4/D4 was not increased. Experiments with cell lysates demonstrated that intracellular formation, rather than secretion, was arrested in hyperosmolar buffers. We conclude that this selective inhibition of mediator production is a unique response of mast cells and basophils to osmotic activation. Although the mechanism of this selective cellular response is not clear, these in vitro observations have important therapeutic and pathophysiologic implications for the airway response to hyperosmolar stimuli.