Our goal was to investigate the role of Ca2+ entry in regulating Cl- current (ICl) in smooth muscle cells from canine trachealis. When studies were done using the perforated patch configuration, depolarization elicited a dihydropyridine-sensitive Ca2+ current (ICa), followed in many cells by a sustained current. This sustained current reversed direction close to the Cl- equilibrium potential, consistent with its representing ICl. The ICl was also apparent as slowly deactivating tail currents seen upon repolarization to negative potentials. The Cl- channel blocker niflumic acid abolished both the sustained and tail currents, without affecting ICa. Several observations indicated that the ICl was dependent on Ca2+ entry. ICl was increased in magnitude when Ca2+ influx was augmented [by prolonging the depolarization or using BAY K 8644 or acetylcholine (ACh)] and decreased in magnitude when Ca2+ influx was reduced (using nifedipine). Based on these findings, we conclude that depolarization causes Ca2+ entry, with resultant elevation of cytosolic free Ca2+ concentration leading to activation of ICl (ICl(Ca)). We investigated whether Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was involved in activation of ICl(Ca), by depleting intracellular stores of Ca2+ using cyclopiazonic acid to block the sarcoplasmic Ca(2+)-adenosinetriphosphatase and repeated stimulation with ACh. In such Ca(2+)-depleted cells, depolarization-mediated Ca2+ entry continued to activate ICl(Ca), suggesting that Ca(2+)-induced Ca2+ release was not required for its activation. We conclude that Ca2+ entry can activate Cl- channels in tracheal smooth muscle. This represents a positive-feedback system, which would promote excitation and contraction of airway muscle.