Amlexanox markedly inhibits histamine release from rat mast cells. To clarify the mechanism of this inhibition, we investigated the effect of amlexanox on cAMP content, which, when increased, inhibits histamine release in rat peritoneal mast cells. At concentrations of 10^––8––10^––6M, amlexanox or isoproterenol increased the cAMP content of mast cells over that of control cells about 2-fold. When the mast cells were incubated with 10^––8, 10^––7 and 10^––6M of amlexanox combined with 10^––7M isoproterenol, the cAMP contents were synergistically increased 15-, 60- and 88-fold, respectively. 3-Isobutyl-1-methylxanthine (IBMX) at 10^––6––10^––4M increased the cAMP content 1.7––3.8-fold, and a combination of 10^––4M IBMX and 10^––7M isoproterenol synergistically increased the cAMP content 41-fold. A combination of amlexanox and IBMX synergistically increased the cAMP content 19-fold. The increase in cAMP content, when amlexanox and isoproterenol were combined, was transient; it peaked at 0.5 min after the drugs were administered, then decreased to 20–30% of the peak value about 2 min later. Pretreatment of mast cells with amlexanox reduced the effect of the combination of amlexanox and isoproterenol, indicating tachyphylaxis; pretreatment with IBMX had no such effect. The cAMP content of macrophages was also increased by amlexanox, but when combined with isoproterenol or PGE₂, the effect was additive. Amlexanox inhibited cAMP phosphodiesterase in rat mast cells; its IC₅₀ value was 1.4 × 10^––5M, and its inhibitory activity was half that of IBMX. These results suggest that amlexanox inhibits histamine release by augmenting the cAMP content in mast cells, and that this augmentation is caused by enhancing the cAMP generation and, additively, by inhibiting cAMP phosphodiesterase.