Secretion of Cl- by epithelial cells is fundamental to the processes of fluid and electrolyte transport by epithelia such as those of the airways, sweat-ducts, and gastrointestinal tract. In the present study, we show that a novel Cl- channel protein, immunoaffinity purified from bovine tracheal apical membrane vesicles, is sensitive to phosphorylation by Ca2+/calmodulin protein kinase II (CaMK II). The channel protein, which migrates with an M(r) of 140,000 under nonreducing conditions, is phosphorylated in vitro by CaMK II in a Ca2+- and calmodulin-dependent manner. When reconstituted into planar lipid bilayers, the protein behaves as an anion-selective, 4,4'-diisothiocyanostilbene- and dithiothreitol-sensitive channel. The open probability of this channel is significantly increased by Ca2+ alone but only at levels of Ca2+ (5-10 μM) that lie outside the physiological range. Addition of CaMK II to the presumptive cytoplasmic side of the bilayer in the presence of ATP and calmodulin dramatically increased the sensitivity of the channel to free Ca2+, shifting the dose-response curve for Ca2+-dependent channel activation to lower [Ca2+](i), the maximum increase in channel P(o) occurring between 0.6 and 1 μM. The addition of kinase in the absence of ATP or calmodulin or the addition of ATP or calmodulin in the absence of kinase was without effect on channel P(o). Increasing [Ca2+] above 1 μM decreased channel mean current, causing a flickery block that was maximal at 2 μM. Increasing [Ca2+] as high as 10 μM in the presence of kinase did not further alter channel behavior. In contrast to CaMK II, the addition of the catalytic subunit of protein kinase A either alone or together with ATP had no effect on channel P(o). These observations suggest that a novel Ca2+-sensitive anion channel isolated from bovine airway epithelium is regulated by CaMK II phosphorylation.