Background: Patients with acute respiratory distress syndrome who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. The release of endogenous catecholamines associated with shock or the administration of β2-adrenergic receptor (β2AR) agonists enhances AFC via a 3′-5′-cyclic adenosine monophosphate-dependent mechanism. The authors have previously reported that transforming growth factor-β1 (TGF-β1) and interleukin-8 (IL-8), two major mediators of alveolar inflammation associated with the early phase of acute respiratory distress syndrome, inhibit AFC upregulation by β2AR agonists via a phosphoinositol-3-kinase (PI3K)-dependent mechanism. However, whether TGF-β1 and IL-8 cause an additive or synergistic inhibition of AFC is unclear. Thus, the central hypothesis of the study was to determine whether they synergistically inhibit the β2AR-stimulated AFC by activating two different isoforms of PI3K. Methods: The effects of TGF-β1 or IL-8 on β2AR agonist-stimulated net alveolar fluid transport were studied using short-circuit current studies. Molecular pathways of inhibition were confirmed by pharmacologic inhibitors and Western blotting of p-Akt, G-protein-coupled receptor kinase 2, protein kinase C-ζ, and phospho-β2AR. Finally, our observations were confirmed by an in vivo model of AFC. Results: Combined exposure to TGF-β1 and IL-8/cytokine-induced neutrophil chemoattractant-1 caused synergistic inhibition of β2AR agonist-stimulated vectorial Cl- across alveolar epithelial type II cells (n = 12 in each group). This effect was explained by activation of different isoforms of PI3K by TGF-β1 and IL-8/cytokine-induced neutrophil chemoattractant-1 (n = 12 in each group). Furthermore, the inhibitory effect of TGF-β1 on 3′-5′-cyclic adenosine monophosphate-stimulated alveolar epithelial fluid transport required the presence of IL-8/cytokine-induced neutrophil chemoattractant-1 (n = 12 in each group). Inhibition of cytokine-induced neutrophil chemoattractant-1 prevented TGF-β1-mediated heterologous β2AR downregulation and restored physiologic β2AR agonist-stimulated AFC in rats (n = 6 in each group). Conclusions: TGF-β1 and IL-8 have a synergistic inhibitory effect on β2AR-mediated stimulation of pulmonary edema removal by the alveolar epithelium. This result may, in part, explain why a large proportion of the patients with acute respiratory distress syndrome have impaired AFC.