In airway smooth muscle cells, interleukin (IL)-4 inhibited both carbachol-and caffeine-induced calcium mobilization from the sarcoplasmic reticulum (SR). Because of the known signaling pathways for IL-4 and importance of calcium uptake in maintaining SR calcium stores shared by agonists and caffeine, it was hypothesized that this rapid inhibitory effect might depend on phosphatidylinositol 3-kinase (PI3K) and on inhibition of calcium uptake by the SR. Enzyme-dispersed bovine trachealis cells were loaded with Fura-2/acetoxymethyl ester, and changes in cytosolic calcium were imaged in single cells. Cells were pretreated with inhibitors of PI3K, either wortmannin (100 nM), LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (50 M), or deguelin (100 nM). Calcium transients in response to carbachol (10 M) were significantly decreased to 0.34 Ϯ 0.10 of control after 20-min treatment with IL-4 but were 1.10 Ϯ 0.26 and 1.08 Ϯ 0.23 when wortmannin or deguelin, respectively, was added along with IL-4. LY294002 alone had nonspecific effects on transients. In other experiments, cyclopiazonic acid (CPA) (5 M), an inhibitor of SR calcium uptake, decreased carbachol-stimulated transients within 4 min to 0.83 Ϯ 0.08 of control (n ϭ 6). However, for cells treated with IL-4 (50 ng/ml) plus CPA, transients decreased significantly more, to only 0.51 Ϯ 0.05 (n ϭ 6; p Ͻ 0.05). Longer exposures to IL-4 and a higher concentration of CPA (30 M) gave similar results. It was concluded that IL-4 did not inhibit transients in the presence of PI3K antagonists but that it did in the presence of CPA. This suggested that IL-4 inhibited calcium transients by mechanisms dependent upon a wortmannin-sensitive PI3K but not by inhibition of calcium uptake into the SR.T helper-2 cytokines, including IL-4 and the closely related cytokine IL-13, have important and complex effects on airway inflammation and airway cells (Brusselle et al