In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to guinea-pig gastric myocytes, which were held at -20 mV using the whole-cell patch-clamp method. ICCh was suppressed by nicardipine or removal of Ca2+ from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7[N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of ICCh. However, @-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5'-O-(3-thiotriphosphate) (GTP¿gammaS] 0.2 mM) in the pipette solution. This GTP[gammaS]-induced cationic current was still markedly suppressed by the Ca2+-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca2+ channels as well as the effects on ICCh. These data suggest that multiple Ca2+-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca2+/calmodulin-dependent pathway would be one of them.