Acetylcholine (ACh) in low doses (0.1-1 microM) reversibly inhibits voltage-dependent activation of the "pacemaker" current, if, in isolated sino-atrial node cells. This action is brought about by a negatively-directed shift of the current activation curve, opposite to that due to catecholamines on the same current. The if inhibition is antagonized by atropine, indicating the involvement of muscarinic receptors. In cells incubated in pertussis toxin-containing solutions, if does not respond to ACh, suggesting that G-proteins mediate the ACh-induced if depression. Further, ACh can inhibit if following catecholamine-induced stimulation, but has a negligible effect on if stimulated by forskolin, a direct activator of adenylate-cyclase. Our results indicate that ACh acts on if by inhibiting basal adenylate-cyclase activity.