We examined the activation and Ca2+ permeation of nonselective cation channels in voltage-clamped (nystatin), fura 2-loaded equine tracheal myocytes at 35 degrees C. Methacholine (50 microM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) and a biphasic inward current consisting of a large, rapidly inactivating Ca(2+)-activated Cl current [ICl(Ca)] and a smaller, sustained nonselective cation current (Icat) ICl(Ca) but not Icat was activated by caffeine. Neither Icat nor the sustained rise in [Ca2+]i was blocked by nisoldipine, whereas both were rapidly blocked by Ni2+; Icat was determined to be Ca2+ permeant, since 1) a sustained elevation of [Ca2+]i occurred when Icat was activated, and blockade of Icat produced a rapid decline in [Ca2+]i; 2) increasing extracellular Ca2+ during Icat increased [Ca2+]i; 3) 110 mM extracellular Ca2+ shifted the reversal potential of Icat to 12 mV (Ca(2+)-to-Cs+ permeability ratio = 3.6); and 4) instantaneous voltage-clamp steps to negative potentials during Icat increased the current and [Ca2+]i, whereas depolarizing steps decreased the current and [Ca2+]i. The fraction of Icat carried by Ca2+ under physiological conditions was estimated to be 14% at -60 mV.