Background: Although the existence of functional β₂-adrenoceptor on eosinophils has been reported, the effects of desensitization of β₂-adrenoceptors on eosinophils have not been well documented. Objective: The effects of desensitization of β₂-adrenoceptors on the degranulation of guinea pig eosinophils were investigated. Methods: Guinea pig eosinophils were stimulated with the calcium ionophore A23187, and eosinophil peroxidase (EPO) release was determined. Changes in intracellular cyclic adenosine monophosphate (cAMP) levels were also measured. Results: A23187-induced EPO release from guinea pig eosinophils was inhibited in a concentration-dependent manner by pretreatment for 5 minutes with fenoterol, clenbuterol, and salbutamol. Such effects of β₂-agonists were abolished by pretreatment with KT5720, an inhibitor of protein kinase A. Desensitization of the inhibitory effects of β₂-agonists was observed when the incubation time was prolonged. Fenoterol (10^–6 mol/L) induced almost complete desensitization after 120 minutes of incubation, whereas clenbuterol did not bring about significant desensitization. The inhibitory effects of fenoterol and clenbuterol on A23187-induced EPO release were correlated with increases in the intracellular cAMP levels evoked by either compound. After incubation of eosinophils with 10^–6 mol/L fenoterol for 120 minutes to induce complete desensitization of β₂-adrenoceptors, the inhibitory effects of theophylline and rolipram were increased by about 100-fold in the desensitized cells, although the effects of forskolin and dibutyryl cAMP were not affected by β₂-adrenoceptor desensitization. Conclusions: Prolonged incubation with β₂-agonists induced desensitization of β₂-adrenoceptors. Also, we postulated that hypersensitization of phosphodiesterase to its inhibitors occurs in β₂-adrenoceptor–desensitized guinea pig eosinophils. (J Allergy Clin Immunol 2000;106:896-903.)