Passive depletion of internal Ca(2+) stores in airway smooth muscle (ASM) activates nonselective cation channels (NSCCs) that mediate capacitative Ca(2+) entry. However, the single channel properties of these cation channels have yet to be resolved and their regulation by cytosolic Ca(2+) levels ([Ca(2+)](i)) still remains unclear. NSCC currents and changes in [Ca(2+)](i) during passive depletion of internal Ca(2+) stores were monitored in isolated bovine tracheal myocytes. Loading cells with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) to reduce [Ca(2+)](i) and thereby deplete the store augmented a basal Gd(3+)- and La(3+)-sensitive, Ca(2+)-permeable NSCC current. This current mimics that which is evoked by store depletion using the sarcoplasmic reticulum Ca(2+) pump inhibitor cyclopiazonic acid (which concurrently and transiently elevates [Ca(2+)](i)). Both interventions activated an approximately 25-pS NSCC with properties identical to both spontaneous (basal) and BAPTA-AM-evoked single channel currents. In summary, the present study provides novel evidence that a lanthanide-sensitive, 25-pS nonselective cation channel underlies both basal and store depletion-evoked membrane currents in airway smooth muscle and that this conductance likely contributes to the regulation of resting [Ca(2+)](i) and capacitative Ca(2+) entry.