The effects of acetylcholine on the frequency-force relationship in isolated atria of the dog were investigated in spontaneously beating and electrically paced preparations, which were suspended in a bath perfused with arterial blood from a carotid artery of a heparinized donor dog. 2. The developed tension at low frequencies of stimulation was less suppressed than at higher frequencies by an infusion of a relatively low dose level (1-2 microgram/min) of acetylcholine. At higher doses (4-10 microgram/min), acetylcholine suppressed the tension development at any given frequency.