The adenosine agonist 5-(N-ethylcarboxamido)adenosine (NECA) induces vasodilation in the pulmonary circulation via A2-adenosine-receptor activation. We addressed whether prolonged treatment with NECA desensitizes in A2-adenosine- receptor function in isolated lung and pulmonary artery smooth muscle cells (PASMC). In lung microcirculation preconstricted with a hypoxic gas, initial administration of NECA caused a 57% vasodilatory response after 3-4 min. Readministration of NECA after 45 min resulted in minimal vasodilation. The highest accumulation of PASMC cAMP occurred 3-5 min after NECA, coincident with NECA-induced vasodilation. In PASMCs treated with NECA for 45 min, cAMP did not increase. Isoproterenol- and indolidan-induced vasodilation remained intact in NECA-desensitized lungs. In NECA-desensitized PASMCs, isoproterenol-induced cAMP accumulation was decreased, suggesting a common mechanism of desensitization. cAMP accumulation was decreased in cholera toxin-treated NECA-desensitized PASMCs compared with cholera toxin-treated control PASMCs, demonstrating that Gsalpha-adenylyl cyclase signaling contributes to desensitization. The A2a-adenosine-receptor agonist CGS-21680C neither increased cAMP accumulation in PASMCs nor attenuated NECA-induced vasodilation. These data support that the A2b-adenosine receptor is responsible for pulmonary vasodilation and desensitization through mechanisms(s) involving Gsalpha-adenylyl cyclase signaling.