Acetylcholine (ACh) increases the amplitude and duration of colonic electrical slow waves. This suggests that ACh either increases an inward current or suppresses an outward current. The latter hypothesis was tested in whole cell voltage-clamp experiments performed on freshly dispersed smooth muscle cells from canine proximal colon. Addition of ACh (10(-5) M) to solutions bathing cells reduced time-dependent outward currents elicited by depolarizing test pulses in the range of -45 to +30 mV. Analysis of tail currents showed that ACh caused a 10- to 15-mV positive shift in voltage-dependent activation. When cells were pretreated with 10(-6) M nifedipine to abolish the Ca2+-dependent component of the outward current, the reduction of outward current by ACh was blocked. Single-channel experiments were performed to determine whether ACh had a direct effect on Ca2+-activated K channels. ACh, 10(-5) M, added to bath and pipette solutions caused a positive shift in voltage-dependent activation in on-cell experiments. This effect of ACh on Ca2+-activated K channels provides a mechanism for the effects of muscarinic, excitatory stimulation of circular muscle of the colon.