Single micro-electrode voltage-clamp and current-clamp techniques were used to study cholinergic responses in single freshly isolated gastric smooth muscle cells from the toad Bufo marinus. Acetylcholine (ACh) or muscarine caused membrane depolarization, which sometimes gave rise to action potentials and contractions. The agonist-induced depolarization is due to the suppression of a voltage-dependent K+ conductance, a conclusion based on the following observations. Depolarization was accompanied by an apparent membrane conductance decrease, seen as the increased size of voltage deflexions in response to constant current pulses. The conductance decrease was confirmed under voltage clamp, where current deflexions in response to constant voltage jumps were smaller in the presence of cholinergic agonists. Muscarine induced net inward currents at potentials positive to the K+ equilibrium potential (EK), and net outward currents at potentials negative to EK. In experiments where external K+ concentration ([K+]o) ranged from 20 to 90 mM the reversal potentials shifted 58 mV positive per tenfold elevation of [K+]o, as expected for a K+ current. The steady-state current-voltage relationship revealed that the K+ current inhibited by muscarine was larger at more positive potentials than expected from driving force considerations alone. Therefore, the underlying conductance suppressed by cholinergic agonists was voltage dependent, with almost complete deactivation at potentials more negative than approximately -70 mV and exhibiting a sigmoidal activation curve upon depolarization. The deactivation of this voltage-dependent K+ conductance caused slow current relaxations to occur in response to hyperpolarizing voltage commands from depolarized holding potentials. In experiments where [K+]o ranged from 3 to 30 mM, these current relaxations reversed direction at potentials near EK and the reversal potential shifted 52 mV positive per tenfold elevation of [K+]o, indicating that K ions carry most of the charge. The current relaxations that occurred in response to hyperpolarizing voltage commands were suppressed by ACh, muscarine and oxotremorine. The effects of muscarine persisted in nominally Ca2+-free solutions containing Mn2+. Ba2+ mimicked the effects of muscarinic agonists. Thus, isolated smooth muscle cells exhibit a K+ current resembling the M-current of sympathetic and other neurones, which is reversibly suppressed by cholinergic agonists. The existence of a cholinergic K+ conductance decrease is of interest because it has not previously been demonstrated in smooth muscle.