The mIMCD-3 cell line, developed from simian virus 40 transformed mice, was grown on coverslips for single-channel analysis of the apical membrane. An amiloride-sensitive nonselective cation channel (NCATP) was demonstrated that occurred predominantly in excised patches. The selectivity sequence for NCATP was NH4+ = Na+ = K+ = Li+ = Rb+ > Cl-. The single-channel conductance was 24 pS and nonrectifying in 140 mM KCl or NaCl solutions. NCATP was not permeable to barium from the extracellular side. NCATP was not voltage gated but was activated spontaneously upon patch excision or after applying negative pressure (20-40 mmHg) to an excised patch in bath solutions containing 1 microM Ca2+ [mean number of open channels (NPl) = 1.45]. NCATP was not activated when excised into a bath solution in which Ca2+ was reduced to 100 nM. The open probability of NCATP was reduced by 68% when 2 mM ATP was added to the intracellular side of an excised patch but was unaffected when 0.1 mM 8-bromoguanosine 3',5'-cyclic monophosphate was added to the intracellular side. In cell-attached patches, NCATP was activated upon response to a hyposmotic (210 mosmol/kgH2O) bathing solution containing 0.5 mM Ca2+ (NPo = 0.35). These results show that the mIMCD-3 cell line contains a volume-sensitive nonselective cation channel that is modulated by ATP and calcium but not guanosine 3',5'-cyclic monophosphate. It is postulated that NCATP may act to initiate the volume regulatory response in IMCD cells.