Isoproterenol (Iso) infusion for 48 h in rats decreases the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). An impairment in protein kinase A (PKA) function appears to be critical in producing the desensitized ALC response. To test this hypothesis, we used a novel protein delivery reagent (Chariot, Active Motif) to deliver either the PKA catalytic subunit or the PKA holoenzyme to the distal lung epithelium of Iso-infused rats (400 microg.kg(-1).h(-1), 48 h). After this infusion, ALC was measured by mass balance over 2 h. ALC in Iso-infused rats was 27.9% (SD 5.8) of instilled volume absorbed. Delivery of the catalytic PKA subunit to Iso-infused rats increased ALC to 47.7% (SD 8.9) (P < 0.05). ALC in Iso-infused rats delivered the inactive PKA holoenzyme [29.6% (SD 2.5)] was not increased above baseline values. Subsequent holoenzyme activation by intravenous infusion of the stable cAMP analog Sp-8-Bromo-cAMPS increased ALC to 41.7% (SD 8.8) (P < 0.05). Immunohistochemical localization of Chariot-delivered PKA revealed staining in the alveolar and distal airway epithelium. These data indicate that protein delivery reagents can be used to rapidly deliver biologically active proteins to the distal lung epithelium and that PKA desensitization may be an important rate-limiting event in the development of Iso-induced desensitization of the alveolar epithelial beta-AR signaling pathway.